Methods of using interleukin-10 for treating solid tumors

ABSTRACT

Methods of treating subjects having a disease or disorder responsive to IL-10, including methods of administration and dosing regimens associated therewith, are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit of U.S. provisional applicationSer. No. 61/813,563, filed Apr. 18, 2013, which application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of using IL-10 and related agents inthe treatment or prevention of a diverse array of diseases anddisorders.

INTRODUCTION

The cytokine interleukin-10 (IL-10) is a pleiotropic cytokine thatregulates multiple immune responses through actions on T cells, B cells,macrophages, and antigen presenting cells (APC). IL-10 may suppressimmune responses by inhibiting expression of IL-1α, IL-1β, IL-6, IL-8,TNF-α, GM-CSF and G-CSF in activated monocytes and activatedmacrophages, and it also suppresses IFN-γ production by NK cells.Although IL-10 is predominantly expressed in macrophages, expression hasalso been detected in activated T cells, B cells, mast cells, andmonocytes. In addition to suppressing immune responses, IL-10 exhibitsimmuno-stimulatory properties, including stimulating the proliferationof IL-2- and IL-4-treated thymocytes, enhancing the viability of Bcells, and stimulating the expression of MHC class II.

Human IL-10 is a homodimer that becomes biologically inactive upondisruption of the non-covalent interactions between the two monomersubunits. Data obtained from the published crystal structure of IL-10indicates that the functional dimer exhibits certain similarities toIFN-γ (Zdanov et al, (1995) Structure (Lond) 3:591-601).

As a result of its pleiotropic activity, IL-10 has been linked to abroad range of diseases, disorders and conditions, includinginflammatory conditions, immune-related disorders, fibrotic disordersand cancer. Clinical and pre-clinical evaluations with IL-10 for anumber of such diseases, disorders and conditions have solidified itstherapeutic potential. Moreover, pegylated IL-10 has been shown to bemore efficacious than non-pegylated IL-10 in certain therapeuticsettings.

In view of the prevalence and severity of IL-10-associated diseases,disorders and conditions, novel dosing regimens and parameters thatoptimize efficacy, patient tolerance and the like would be of tremendousvalue in furthering the therapeutic usefulness of IL-10 and pegylatedIL-10, and agents related thereto.

SUMMARY

The present disclosure contemplates methods of using IL-10, modified(e.g., pegylated) IL-10, and associated agents described herein, andcompositions thereof, to treat and/or prevent various diseases,disorders and conditions, and/or the symptoms thereof. Moreparticularly, the present disclosure relates to optimized dosingparameters to achieve and maintain efficacy in the treatment and/orprevention of various diseases, disorders and conditions in a subject,while minimizing the adverse effects associated therewith. As set forthe in detail hereafter, such optimization of dosing parametersinvolves, for example, the assessment of pharmacokinetic andpharmacodynamic parameters associated with absorption, distribution,metabolism, and excretion (“ADME”), taking into consideration the routeof administration and other factors. It is understood that, unlessindicated otherwise herein, terms related to ADME and other parametersare intended to have their ordinary accepted meanings in the relevantscientific fields. By way of example, the terms “serum half-life” or“t_(1/2),” refer to elimination half-life (i.e., the time at which theserum concentration of an agent has reached one-half of its initial ormaximum value).

According to the methods described herein, the disease, disorder orcondition, and/or symptoms thereof, may be a proliferative disorder,such as cancer or a cancer-related disorder, or a fibrotic disorder,such as cirrhosis, NASH and NAFLD. Though not limited to particularcancers, the cancer may be a solid tumor, including tumors associatedwith colon cancer, melanoma, and squamous cell carcinoma, or it may be ahematological disorder.

In other embodiments, the disease, disorder or condition is a viraldisorder, including, but not limited to, human immunodeficiency virus,hepatitis B or C virus or cytomegalovirus. In still further embodiments,the disease, disorder or condition is an immune or inflammatorydisorder, which may be acute or chronic. Examples of immune andinflammatory disorders include inflammatory bowel disease, psoriasis,rheumatoid arthritis, multiple sclerosis, and Alzheimer's disease.

In particular embodiments, the disease, disorder or condition is acardiovascular disorder, including atherosclerosis. The subject having acardiovascular disorder may have elevated cholesterol.

In still further embodiments, the disease, disorder or condition isthrombosis or a thrombotic condition.

As discussed further hereafter, human IL-10 is a homodimer and eachmonomer comprises 178 amino acids, the first 18 of which comprise asignal peptide. Particular embodiments of the present disclosurecomprise mature human IL-10 polypeptides lacking the signal peptide(see, e.g., U.S. Pat. No. 6,217,857), or mature human PEG-IL-10. Infurther particular embodiments, the IL-10 agent is a variant of maturehuman IL-10. The variant may exhibit activity less than, comparable to,or greater than the activity of mature human IL-10; in certainembodiments the activity is comparable to or greater than the activityof mature human IL-10.

Certain embodiments of the present disclosure contemplate modificationof IL-10 in order to enhance one or more properties (e.g.,pharmacokinetic parameters, efficacy, etc.). In particular embodiments,IL-10 is modified by, for example, pegylation, glycosylation, albumin(e.g., human serum albumin (HSA)) conjugation, and hesylation. Infurther embodiments, modification of IL-10 does not result in atherapeutically relevant, detrimental effect on immunogenicity, and instill further embodiments modified IL-10 is less immunogenic thanunmodified IL-10. The terms “IL-10”, “IL-10 polypeptide(s),” “agent(s)”and the like are intended to be construed broadly and include, forexample, human and non-human IL-10-related polypeptides, includinghomologs, variants (including muteins), and fragments thereof, as wellas IL-10 polypeptides having, for example, a leader sequence (e.g., thesignal peptide), and modified versions of the foregoing. In furtherparticular embodiments, the terms “IL-10”, “IL-10 polypeptide(s),“agent(s)” are agonists. Particular embodiments relate to pegylatedIL-10, which is also referred to herein as “PEG-IL-10”. The presentdisclosure also contemplates nucleic acid molecules encoding theforegoing.

Particular embodiments of the present disclosure relate to methods oftreating or preventing a disease, disorder or condition in a subject,comprising administering to the subject a therapeutically effectiveamount of an IL-10 agent, wherein the amount is sufficient to achieve amean IL-10 serum trough concentration of at least 0.1 ng/mL. The methodsof treating or preventing may be mediated by CD8+ T cells.

Other embodiments relate to methods of treating or preventing a disease,disorder or condition in a subject (e.g., a human), comprisingadministering to the subject a therapeutically effective amount of anIL-10 agent, wherein the amount is sufficient to maintain a mean IL-10serum trough concentration over a period of time, wherein the mean IL-10serum trough concentration is at least 0.1 ng/mL, and wherein the meanIL-10 serum trough concentration is maintained for at least 90% of theperiod of time. In particular embodiments of the present disclosure, themean IL-10 serum trough concentration is at least 0.2 ng/mL, at least0.3 ng/mL, and least 0.4 ng/mL, at least 0.5 ng/mL, at least 0.6 ng/mL,at least 0.7 ng/mL, at least 0.8 ng/mL, at least 0.9 ng/mL, at least 1ng/mL, at least 1.2 ng/mL, at least 1.25 ng/mL, at least 1.3 ng/mL, atleast 1.4 ng/mL, at least 1.5 ng/mL, at least 1.6 ng/mL, at least 1.7ng/mL, at least 1.8 ng/mL, at least 1.85 ng/mL, at least 1.9 ng/mL, atleast 1.95 ng/mL, at least 1.97 ng/mL, and least 1.98 ng/mL, at least1.99 ng/mL, at least 2.0 ng/mL or greater than 2 ng/mL.

In further embodiments, the period of time is at least 12 hours, atleast 24 hours, at least 48 hours, at least 72 hours, at least 1 week,at least 2 weeks, at least 3 weeks, at least 1 month, at least 6 weeks,at least 2 months, at least 3 months, or greater than 3 months.

In particular embodiments of the present disclosure, the mean IL-10serum trough concentration is maintained for at least 85% of the periodof time, at least 90%, at least 95%, at least 98%, at least 99% or 100%of the period of time.

It is envisaged that a dosing regimen sufficient to maintain a desiredsteady state serum trough concentration (e.g., 0.1 ng/mL or 2 ng/mL) mayresult in an initial serum trough concentration that is higher than thedesired steady state serum trough concentration. Because of thepharmacodynamic and pharmacokinetic characteristics of IL-10 in amammalian subject, an initial trough concentration (achieved, forexample, through the administration of one or more loading dosesfollowed by a series of maintenance doses) gradually but continuallydecreases over a period of time even when the dosing parameters (amountand frequency) are kept constant. After that period of time, the gradualbut continual decrease ends and a steady state serum troughconcentration is maintained.

By way of example, parenteral administration (e.g., SC and IV) of ˜0.1mg/kg/day of an IL-10 agent (e.g., mIL-10) to a mouse (e.g., a C57BL/6mouse) is required to maintain a steady state serum trough concentrationof; for example, 2.0 ng/mL. However, that steady state scrum troughconcentration may not be achieved until approximately 30 days afterinitiation of dosing at 0.1 mg/kg/day (and also after any loadingdose(s)). Rather, after an initial serum trough concentration has beenachieved (e.g., 2.5 ng/mL), that concentration gradually but continuallydecreases over the course of, for example, the approximately 30-dayperiod, after which time the desired steady state serum troughconcentration (e.g., 2.0 ng/mL) is maintained. One of skill in the artwill be able to determine the dose needed to maintain the desired steadystate trough concentration using, for example, ADME and patient-specificparameters.

The present disclosure contemplates methods wherein the IL-10 agent maycomprise at least one modification to form a modified IL-10 agent,wherein the modification does not alter the amino acid sequence of theIL-10 agent. In some embodiments, the modified IL-10 agent is aPEG-IL-10 agent. The PEG-IL-10 agent may comprise at least one PEGmolecule covalently attached to at least one amino acid residue of atleast one subunit of IL-10 or comprise a mixture of mono-pegylated anddi-pegylated IL-10 in other embodiments. The PEG component of thePEG-IL-10 agent may have a molecular mass greater than about 5 kDa,greater than about 10 kDa, greater than about 15 kDa, greater than about20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greaterthan about 50 kDa. In some embodiments, the molecular mass is from about5 kDa to about 10 kDa, from about 5 kDa to about 15 kDa, from about 5kDa to about 20 kDa, from about 10 kDa to about 15 kDa, from about 10kDa to about 20 kDa, from about 10 kDa to about 25 kDa or from about 10kDa to about 30 kDa.

In some embodiments, the modified IL-10 agent comprises at least one Fcfusion molecule, at least one serum albumin (e.g., HSA or BSA), an HSAfusion molecule or an albumin conjugate. In additional embodiments, themodified IL-10 agent is glycosylated, is hesylated, or comprises atleast one albumin binding domain. Some modified IL-10 agents maycomprise more than one type of modification. In particular embodiments,the modification is site-specific. Some embodiments comprise a linker.Modified IL-10 agents are discussed in detail hereafter.

The present disclosure also contemplates the use of gene therapy inconjunction with the teachings herein. For gene therapy uses andmethods, a cell in a subject can be transformed with a nucleic acid thatencodes an IL-10-related polypeptide as set forth herein in vivo.Alternatively, a cell can be transformed in vitro with a transgene orpolynucleotide, and then transplanted into a tissue of subject in orderto effect treatment. In addition, a primary cell isolate or anestablished cell line can be transformed with a transgene orpolynucleotide that encodes an IL-10-related polypeptide, and thenoptionally transplanted into a tissue of a subject.

The present disclosure contemplates methods wherein the IL-10 agent isadministered to the subject at least twice daily, at least once daily,at least once every 48 hours, at least once every 72 hours, at leastonce weekly, at least once every 2 weeks, at least once monthly, atleast once every 2 months, or at least once every 3 months. Someembodiments also comprise administering the IL-10 agent with at leastone additional prophylactic or therapeutic agent, examples of which areset forth hereafter.

The IL-10 agent may be administered by any effective route. In someembodiments, it is administered by parenteral injection, includingsubcutaneous injection.

Particular embodiments of the present disclosure relate topharmaceutical compositions comprising an amount of an IL-10 agent(e.g., a therapeutically effective amount), including those agentsdescribed above, along with one or more pharmaceutically acceptablediluent, carrier or excipient (e.g., an isotonic injection solution).The pharmaceutical composition is generally one that is suitable forhuman administration. Furthermore, in some embodiments thepharmaceutical composition comprises at least one additionalprophylactic or therapeutic agent.

Certain embodiments of the present disclosure contemplate a sterilecontainer that contains one of the above-mentioned pharmaceuticalcompositions and optionally one or more additional components. By way ofexample, but not limitation, the sterile container may be a syringe. Instill further embodiments, the sterile container is one component of akit; the kit may also contain, for example, a second sterile containerthat comprises at least one prophylactic or therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of human and mouse IL-10.

FIG. 2A depicts the concentration of MCP-1 (pg/mL) in PBMCs atincreasing concentrations of IL-10. At concentrations of 1 ng/mL andabove, IL-10 increased the secretion of MCP-1.

FIG. 2B depicts the concentration of MCP-1 (pg/mL) in PBMCs stimulatedwith LPS at increasing concentrations of IL-10. IL-10 is an inhibitor ofLPS-mediated activation of PBMCs, and the addition of IL-10 atconcentrations of 1 ng/ml, and above significantly inhibited thesecretion of MCP-1.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology such as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Further,the dates of publication provided may be different from the actualpublication dates, which may need to be independently confirmed.

Overview

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousdiseases, disorders and conditions, and/or the symptoms thereof. Incertain aspects of the present disclosure, such treatment or preventionis effected by utilizing particular dosing parameters. In someembodiments the agents are administered so as to achieve a serum troughconcentration that is optimized for treating, for example, inflammatory-and immune-related disorders, fibrotic disorders, cancer andcancer-related disorders, or cardiovascular disorders (e.g.,atherosclerosis).

In some embodiments of the present disclosure, a subject having, or atrisk of having, a disease or disorder treatable by an IL-10 agent (e.g.,an IL-10 polypeptide) is administered the IL-10 agent in an amountsufficient to achieve a serum trough concentration greater than about0.1 ng/mL, in certain embodiments the serum trough concentration isgreater than about 1 ng/mL, whereas in other embodiments the serumtrough concentration is greater than about 2 ng/mL.

It should be noted that any reference to “human” in connection with thepolypeptides and nucleic acid molecules of the present disclosure is notmeant to be limiting with respect to the manner in which the polypeptideor nucleic acid is obtained or the source, but rather is only withreference to the sequence as it may correspond to a sequence of anaturally occurring human polypeptide or nucleic acid molecule. Inaddition to the human polypeptides and the nucleic acid molecules whichencode them, the present disclosure contemplates IL-10-relatedpolypeptides and corresponding nucleic acid molecules from otherspecies.

Definitions

Unless otherwise indicated, the following terms are intended to have themeaning set forth below. Other terms are defined elsewhere throughoutthe specification.

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “administration”, “administer” and the like, as they apply to,for example, a subject, cell, tissue, organ, or biological fluid, referto contact of, for example, IL-10 or PEG-IL-10, a nucleic acid (e.g., anucleic acid encoding native human IL-10); a pharmaceutical compositioncomprising the foregoing, or a diagnostic agent to the subject, cell,tissue, organ, or biological fluid. In the context of a cell,administration includes contact (e.g., in vitro or ex vivo) of a reagentto the cell, as well as contact of a reagent to a fluid, where the fluidis in contact with the cell.

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering IL-10 or a pharmaceutical compositioncomprising IL-10) initiated after a disease, disorder or condition, or asymptom thereof, has been diagnosed, observed, and the like so as toeliminate, reduce, suppress, mitigate, or ameliorate, either temporarilyor permanently, at least one of the underlying causes of a disease,disorder, or condition afflicting a subject, or at least one of thesymptoms associated with a disease, disorder, or condition afflicting asubject. Thus, treatment includes inhibiting (e.g., arresting thedevelopment or further development of the disease, disorder or conditionor clinical symptoms association therewith) an active disease. The termsmay also be used in other contexts, such as situations where IL-10 orPEG-IL-10 contacts an IL-10 receptor in, for example, the fluid phase orcolloidal phase.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering IL-10 or a pharmaceuticalcomposition comprising IL-10) initiated in a manner (e.g., prior to theonset of a disease, disorder, condition or symptom thereof) so as toprevent, suppress, inhibit or reduce, either temporarily or permanently,a subject's risk of developing a disease, disorder, condition or thelike (as determined by, for example, the absence of clinical symptoms)or delaying the onset thereof, generally in the context of a subjectpredisposed to having a particular disease, disorder or condition. Incertain instances, the terms also refer to slowing the progression ofthe disease, disorder or condition or inhibiting progression thereof toa harmful or otherwise undesired state.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount capable of having any detectable, positiveeffect on any symptom, aspect, or characteristic of a disease, disorderor condition when administered to the subject. The therapeuticallyeffective amount can be ascertained by measuring relevant physiologicaleffects, and it can be adjusted in connection with the dosing regimenand diagnostic analysis of the subject's condition, and the like. By wayof example, measurement of the amount of inflammatory cytokines producedfollowing administration may be indicative of whether a therapeuticallyeffective amount has been used.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., serumconcentration of IL-10) or subjective parameter (e.g., a subject'sfeeling of well-being).

The term “small molecules” refers to chemical compounds having amolecular weight that is less than about 10 kDa, less than about 2 kDa,or less than about 1 kDa. Small molecules include, but are not limitedto, inorganic molecules, organic molecules, organic molecules containingan inorganic component, molecules comprising a radioactive atom, andsynthetic molecules. Therapeutically, a small molecule may be morepermeable to cells, less susceptible to degradation, and less likely toelicit an immune response than large molecules.

The term “ligand” refers to, for example, a peptide, polypeptide,membrane-associated or membrane-bound molecule, or complex thereof, thatcan act as an agonist or antagonist of a receptor. “Ligand” encompassesnatural and synthetic ligands, e.g., cytokines, cytokine variants,analogs, muteins, and binding compositions derived from antibodies.“Ligand” also encompasses small molecules, e.g., peptide mimetics ofcytokines and peptide mimetics of antibodies. The term also encompassesan agent that is neither an agonist nor antagonist, but that can bind toa receptor without significantly influencing its biological properties,e.g., signaling or adhesion. Moreover, the term includes amembrane-bound ligand that has been changed, e.g., by chemical orrecombinant methods, to a soluble version of the membrane-bound ligand.A ligand or receptor may be entirely intracellular, that is, it mayreside in the cytosol, nucleus, or some other intracellular compartment.The complex of a ligand and receptor is termed a “ligand-receptorcomplex”.

The terms “inhibitors” and “antagonists”, or “activators” and “agonists”refer to inhibitory or activating molecules, respectively, for example,for the activation of, e.g., a ligand, receptor, cofactor, gene, cell,tissue, or organ. Inhibitors are molecules that decrease, block,prevent, delay activation, inactivate, desensitize, or down-regulate,e.g., a gene, protein, ligand, receptor, or cell. Activators aremolecules that increase, activate, facilitate, enhance activation,sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, orcell. An inhibitor may also be defined as a molecule that reduces,blocks, or inactivates a constitutive activity. An “agonist” is amolecule that interacts with a target to cause or promote an increase inthe activation of the target. An “antagonist” is a molecule that opposesthe action(s) of an agonist. An antagonist prevents, reduces, inhibits,or neutralizes the activity of an agonist, and an antagonist can alsoprevent, inhibit, or reduce constitutive activity of a target, e.g., atarget receptor, even where there is no identified agonist.

The terms “modulate”, “modulation” and the like refer to the ability ofa molecule (e.g., an activator or an inhibitor) to increase or decreasethe function or activity of an IL-10 agent (or the nucleic acidmolecules encoding them), either directly or indirectly; or to enhancethe ability of a molecule to produce an effect comparable to that of anIL-10 agent. The term “modulator” is meant to refer broadly to moleculesthat can effect the activities described above. By way of example, amodulator of, e.g., a gene, a receptor, a ligand, or a cell, is amolecule that alters an activity of the gene, receptor, ligand, or cell,where activity can be activated, inhibited, or altered in its regulatoryproperties. A modulator may act alone, or it may use a cofactor, e.g., aprotein, metal ion, or small molecule. The term “modulator” includesagents that operate through the same mechanism of action as IL-10 (i.e.,agents that modulate the same signaling pathway as IL-10 in a manneranalogous thereto) and are capable of eliciting a biological responsecomparable to (or greater than) that of IL-10.

Examples of modulators include small molecule compounds and otherbioorganic molecules. Numerous libraries of small molecule compounds(e.g., combinatorial libraries) are commercially available and can serveas a starting point for identifying a modulator. The skilled artisan isable to develop one or more assays (e.g., biochemical or cell-basedassays) in which such compound libraries can be screened in order toidentify one or more compounds having the desired properties;thereafter, the skilled medicinal chemist is able to optimize such oneor more compounds by, for example, synthesizing and evaluating analogsand derivatives thereof. Synthetic and/or molecular modeling studies canalso be utilized in the identification of an Activator.

The “activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor; to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity; to the modulation of activities ofother molecules, and the like. The term may also refer to activity inmodulating or maintaining cell-to-cell interactions (e.g., adhesion), oractivity in maintaining a structure of a cell (e.g., a cell membrane).“Activity” can also mean specific activity, e.g., [catalyticactivity]/[mg protein], or [immunological activity]/[mg protein],concentration in a biological compartment, or the like. The term“proliferative activity” encompasses an activity that promotes, that isnecessary for, or that is specifically associated with, for example,normal cell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

As used herein, “comparable”, “comparable activity”, “activitycomparable to”, “comparable effect”, “effect comparable to”, and thelike are relative terms that can be viewed quantitatively and/orqualitatively. The meaning of the terms is frequently dependent on thecontext in which they are used. By way of example, two agents that bothactivate a receptor can be viewed as having a comparable effect from aqualitative perspective, but the two agents can be viewed as lacking acomparable effect from a quantitative perspective if one agent is onlyable to achieve 20% of the activity of the other agent as determined inan art-accepted assay (e.g., a dose-response assay) or in anart-accepted animal model. When comparing one result to another result(e.g., one result to a reference standard), “comparable” frequentlymeans that one result deviates from a reference standard by less than35%, by less than 30%, by less than 25%, by less than 20%, by less than15%, by less than 10%, by less than 7%, by less than 5%, by less than4%, by less than 3%, by less than 2%, or by less than 1%. In particularembodiments, one result is comparable to a reference standard if itdeviates by less than 15%, by less than 10%, or by less than 5% from thereference standard. By way of example, but not limitation, the activityor effect may refer to efficacy, stability, solubility, orimmunogenicity.

The term “response,” for example, of a cell, tissue, organ, or organism,encompasses a change in biochemical or physiological behavior, e.g.,concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming. In certaincontexts, the terms “activation”, “stimulation”, and the like refer tocell activation as regulated by internal mechanisms, as well as byexternal or environmental factors; whereas the terms “inhibition”,“down-regulation” and the like refer to the opposite effects.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence; fusion proteins with heterologous andhomologous leader sequences; fusion proteins with or without N-terminusmethionine residues; fusion proteins with immunologically taggedproteins; and the like.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided below:

G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu IIsoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe YTyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R ArginineArg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic AcidAsp S Serine Ser T Threonine Thr

As used herein, the term “variant” encompasses naturally-occurringvariants and non-naturally-occurring variants. Naturally-occurringvariants include homologs (polypeptides and nucleic acids that differ inamino acid or nucleotide sequence, respectively, from one species toanother), and allelic variants (polypeptides and nucleic acids thatdiffer in amino acid or nucleotide sequence, respectively, from oneindividual to another within a species). Non-naturally-occurringvariants include polypeptides and nucleic acids that comprise a changein amino acid or nucleotide sequence, respectively, where the change insequence is artificially introduced (e.g., muteins); for example, thechange is generated in the laboratory by human intervention (“hand ofman”). Thus, herein a “mutein” refers broadly to mutated recombinantproteins that usually carry single or multiple amino acid substitutionsand are frequently derived from cloned genes that have been subjected tosite-directed or random mutagenesis, or from completely synthetic genes.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” anIL-10 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring IL-10 polypeptide or anIL-10-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologs or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it may naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring. “isolated”indicates that the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist) so that a polypeptide of interest is present in a) a greaterconcentration (e.g., at least 3-fold greater, at least 4-fold greater,at least 8-fold greater, at least 64-fold greater, or more) than theconcentration of the polypeptide in the starting sample, such as abiological sample (e.g., a sample in which the polypeptide naturallyoccurs or in which it is present after administration), or b) aconcentration greater than the environment in which the polypeptide wasmade (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “specifically binds” or “selectively binds”, when referring toa ligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated conditions, a specified ligand binds to a particularreceptor and does not bind in a significant amount to other proteinspresent in the sample. The antibody, or binding composition derived fromthe antigen-binding site of an antibody, of the contemplated methodbinds to its antigen, or a variant or mutein thereof, with an affinitythat is at least two-fold greater, at least ten times greater, at least20-times greater, or at least 100-times greater than the affinity withany other antibody, or binding composition derived therefrom. In aparticular embodiment, the antibody will have an affinity that isgreater than about 10⁹ liters/mol, as determined by, e.g., Scatchardanalysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239).

IL-10 and PEG-IL-10

The anti-inflammatory cytokine IL-10, also known as human cytokinesynthesis inhibitory factor (CSIF), is classified as a type(class)-2cytokine, a set of cytokines that includes IL-19, IL-20, IL-22, IL-24(Mda-7), and IL-26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω, and -τ)and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29).

IL-10 is a cytokine with pleiotropic effects in immunoregulation andinflammation. It is produced by mast cells, counteracting theinflammatory effect that these cells have at the site of an allergicreaction. While it is capable of inhibiting the synthesis ofpro-inflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα and GM-CSF,IL-10 is also stimulatory towards certain T cells and mast cells andstimulates B-cell maturation, proliferation and antibody production.IL-10 can block NF-κB activity and is involved in the regulation of theJAK-STAT signaling pathway. It also induces the cytotoxic activity ofCD8+ T-cells and the antibody production of B-cells, and it suppressesmacrophage activity and tumor-promoting inflammation. The regulation ofCD8+ T-cells is dose-dependent, wherein higher doses induce strongercytotoxic responses.

Human IL-10 is a homodimer with a molecular mass of 37 kDa, wherein each18.5 kDa monomer comprises 178 amino acids, the first 18 of whichcomprise a signal peptide, and two pairs of cysteine residues that formtwo intramolecular disulfide bonds. The IL-10 dimer becomes biologicallyinactive upon disruption of the non-covalent interactions between thetwo monomer subunits.

The present disclosure contemplates human IL-10 and murine IL-10, whichexhibit 80% homology, and use thereof. In addition, the scope of thepresent disclosure includes IL-10 orthologs, and modified forms thereof,from other mammalian species, including rat (accession NP_036986.2; GI148747382); cow (accession NP_776513.1; GI 41386772); sheep (accessionNP_001009327.1; GI 57164347); dog (accession ABY86619.1; GI 166244598);and rabbit (accession AAC23839.1; GI 3242896).

As alluded to above, the terms “IL-10”, “IL-10 polypeptide(s), “IL-10agent(s)” and the like are intended to be broadly construed and include,for example, human and non-human IL-10-related polypeptides, includinghomologs, variants (including muteins), and fragments thereof, as wellas IL-10 polypeptides having, for example, a leader sequence (e.g., thesignal peptide), and modified versions of the foregoing. In furtherparticular embodiments, IL-10, IL-10 polypeptide(s), and IL-10 agent(s)are agonists.

The IL-10 receptor, a type 11 cytokine receptor, consists of alpha andbeta subunits, which are also referred to as R1 and R2, respectively.Receptor activation requires binding to both alpha and beta. Onehomodimer of an IL-10 polypeptide binds to alpha and the other homodimerof the same IL-10 polypeptide binds to beta.

The utility of recombinant human IL-10 is frequently limited by itsrelatively short serum half-life, which may be due to, for example,renal clearance, proteolytic degradation and monomerization in the bloodstream. As a result, various approaches have been explored to improvethe pharmacokinetic profile of IL-10 without disrupting its dimericstructure and thus adversely affecting its activity. Pegylation of IL-10results in improvement of certain pharmacokinetic parameters (e.g.,serum half-life) and/or enhancement of activity. For example, particularembodiments of the present disclosure involve methods of optimizing thetreatment of proliferative disorders (e.g., cancer) with PEG-IL-10.

As previously indicated, the present disclosure also contemplates theuse of gene therapy in conjunction with the teachings herein. Genetherapy is effected by delivering genetic material, usually packaged ina vector, to endogenous cells within a subject in order to introducenovel genes, to introduce additional copies of pre-existing genes, toimpair the functioning of existing genes, or to repair existing butnon-functioning genes. Once inside cells, the nucleic acid is expressedby the cell machinery, resulting in the production of the protein ofinterest. In the context of the present disclosure, gene therapy is usedas a therapeutic to deliver nucleic acid that encodes an IL-10 agent foruse in the treatment or prevention of a disease, disorder or conditiondescribed herein.

As alluded to above, for gene therapy uses and methods, a cell in asubject can be transformed with a nucleic acid that encodes anIL-10-related polypeptide as set forth herein in vivo. Alternatively, acell can be transformed in vitro with a transgene or polynucleotide, andthen transplanted into a tissue of subject in order to effect treatment.In addition, a primary cell isolate or an established cell line can betransformed with a transgene or polynucleotide that encodes anIL-10-related polypeptide, and then optionally transplanted into atissue of a subject.

As used herein, the terms “pegylated IL-10” and PEG-IL-10” refer to anIL-10 molecule having one or more polyethylene glycol moleculescovalently attached to at least one amino acid residue of the IL-10protein, generally via a linker, such that the attachment is stable. Theterms “monopegylated IL-10” and “mono-PEG-IL-10” indicate that onepolyethylene glycol molecule is covalently attached to a single aminoacid residue on one subunit of the IL-10 dimer, generally via a linker.In certain embodiments, the PEG-IL-10 used in the present disclosure isa mono-PEG-IL-10 in which one to nine PEG molecules are covalentlyattached via a linker to the alpha amino group of the amino acid residueat the N-terminus of one subunit of the IL-10 dimer. Monopegylation onone IL-10 subunit generally results in a non-homogeneous mixture ofnon-pegylated, monopegylated and dipegylated IL-10 due to subunitshuffling. Moreover, allowing a pegylation reaction to proceed tocompletion will generally result in non-specific and multi-pegylatedIL-10, thus reducing its bioactivity. Thus, particular embodiments ofthe present disclosure comprise the administration of a mixture of mono-and di-pegylated IL-10 produced by the methods described herein (e.g.,the Experimental section).

In particular embodiments, the average molecular weight of the PEGmoiety is between about 5 kDa and about 50 kDa. Although the method orsite of PEG attachment to IL-10 is not critical, in certain embodimentsthe pegylation does not alter, or only minimally alters, the activity ofthe IL-10 agent. In certain embodiments, the increase in half-life isgreater than any decrease in biological activity. The biologicalactivity of PEG-IL-10 is typically measured by assessing the levels ofinflammatory cytokines (e.g., TNF-α or IFN-γ) in the serum of subjectschallenged with a bacterial antigen (lipopolysaccharide (LPS)) andtreated with PEG-IL-10, as described in U.S. Pat. No. 7,052,686.

IL-10 variants can be prepared with various objectives in mind,including increasing serum half-life, reducing an immune responseagainst the IL-10, facilitating purification or preparation, decreasingconversion of IL-10 into its monomeric subunits, improving therapeuticefficacy, and lessening the severity or occurrence of side effectsduring therapeutic use. The amino acid sequence variants are usuallypredetermined variants not found in nature, although some may bepost-translational variants, e.g., glycosylated variants. Any variant ofIL-10 can be used provided it retains a suitable level of IL-10activity. In the tumor context, suitable IL-10 activity includes, forexample, CD8+ T cell infiltration into tumor sites, expression ofinflammatory cytokines such as IFN-γ, IL-4, IL-6, IL-10, and RANK-L,from these infiltrating cells, and increased levels of IFN-γ inbiological samples.

The phrase “conservative amino acid substitution” refers tosubstitutions that preserve the activity of the protein by replacing anamino acid(s) in the protein with an amino acid with a side chain ofsimilar acidity, basicity, charge, polarity, or size of the side chain.Conservative amino acid substitutions generally entail substitution ofamino acid residues within the following groups: 1) L, 1. M, V, F; 2) R,K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Guidance forsubstitutions, insertions, or deletions may be based on alignments ofamino acid sequences of different variant proteins or proteins fromdifferent species. Thus, in addition to any naturally-occurring IL-10polypeptide, the present disclosure contemplates having 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acidsubstitutions, where the substitution is usually a conservative aminoacid substitution.

The present disclosure also contemplates active fragments (e.g.,subsequences) of mature IL-10 containing contiguous amino acid residuesderived from the mature IL-10. The length of contiguous amino acidresidues of a peptide or a polypeptide subsequence varies depending onthe specific naturally-occurring amino acid sequence from which thesubsequence is derived. In general, peptides and polypeptides may befrom about 20 amino acids to about 40 amino acids, from about 40 aminoacids to about 60 amino acids, from about 60 amino acids to about 80amino acids, from about 80 amino acids to about 100 amino acids, fromabout 100 amino acids to about 120 amino acids, from about 120 aminoacids to about 140 amino acids, from about 140 amino acids to about 150amino acids, from about 150 amino acids to about 155 amino acids, fromabout 155 amino acids up to the full-length peptide or polypeptide.

Additionally, IL-10 polypeptides can have a defined sequence identitycompared to a reference sequence over a defined length of contiguousamino acids (e.g., a “comparison window”). Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch. J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman. Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

As an example, a suitable IL-10 polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, amino acid sequence identity to a contiguous stretch offrom about 20 amino acids to about 40 amino acids, from about 40 aminoacids to about 60 amino acids, from about 60 amino acids to about 80amino acids, from about 80 amino acids to about 100 amino acids, fromabout 100 amino acids to about 120 amino acids, from about 120 aminoacids to about 140 amino acids, from about 140 amino acids to about 150amino acids, from about 150 amino acids to about 155 amino acids, fromabout 155 amino acids up to the full-length peptide or polypeptide.

As discussed further below, the IL-10 polypeptides may be isolated froma natural source (e.g., an environment other than itsnaturally-occurring environment) and may also be recombinantly made(e.g., in a genetically modified host cell such as bacteria, yeast,Pichia, insect cells, and the like), where the genetically modified hostcell is modified with a nucleic acid comprising a nucleotide sequenceencoding the polypeptide. The IL-10 polypeptides may also besynthetically produced (e.g., by cell-free chemical synthesis).

Nucleic acid molecules encoding the IL-10 agents are contemplated by thepresent disclosure, including their naturally-occurring andnon-naturally occurring isoforms, allelic variants and splice variants.The present disclosure also encompasses nucleic acid sequences that varyin one or more bases from a naturally-occurring DNA sequence but stilltranslate into an amino acid sequence that corresponds to an IL-10polypeptide due to degeneracy of the genetic code.

IL-10 Serum Concentration

The blood plasma levels of IL-10 in the methods described herein may becharacterized in several manners, including: (1) a mean IL-10 serumtrough concentration above some specified level or in a range of levels;(2) a mean IL-10 serum trough concentration above some specified levelfor some amount of time; (3) a steady state IL-10 serum concentrationlevel above or below some specified level or in a range of levels; or(4) a C_(max) of the concentration profile above or below some specifiedlevel or in some range of levels. As set forth herein, mean serum troughIL-10 concentrations have been found to be of particular import forefficacy in certain indications.

In some embodiments of the present disclosure, blood plasma levelconcentration profiles that may be produced include: a mean IL-10 serumtrough concentration of greater than about 0.1 ng/mL, greater than about0.15 ng/mL, greater than about 0.2 ng/mL, greater than about 0.25 ng/mL,greater than about 0.3 ng/mL, greater than about 0.35 ng/mL, greaterthan about 0.4 ng/mL, greater than about 0.45 ng/mL, greater than about0.5 ng/mL, greater than about 0.55 ng/mL, greater than about 0.6 ng/mL,greater than about 0.65 ng/mL, greater than about 0.7 ng/mL, greaterthan about 0.75 ng/mL, greater than about 0.8 ng/mL, greater than about0.85 ng/mL, greater than about 0.9 ng/mL, greater than about 0.95 ng/mL,greater than about 1.0 ng/mL, greater than about 1.1 ng/mL, greater thanabout 1.2 ng/mL, greater than about 1.3 ng/mL, greater than about 1.4ng/mL, greater than about 1.5 ng/mL, greater than about 1.6 ng/mL,greater than about 1.7 ng/mL, greater than about 1.8 ng/mL, greater thanabout 1.9 ng/mL, greater than about 2.0 ng/mL, greater than about 2.1ng/mL, greater than about 2.2 ng/mL, greater than about 2.3 ng/mL,greater than about 2.4 ng/mL, greater than about 2.5 ng/mL, greater thanabout 2.75 ng/mL, or greater than about 3.0 ng/mL.

In particular embodiments directed to the treatment or prevention ofcancer-related-diseases, disorders or condition, therapy is optimized byachieving a mean IL-10 serum trough concentration of greater than about0.5 ng/mL, greater than about 0.55 ng/mL, greater than about 0.6 ng/mL,greater than about 0.65 ng/mL, greater than about 0.7 ng/mL, greaterthan about 0.75 ng/mL, greater than about 0.8 ng/mL, greater than about0.85 ng/mL, greater than about 0.9 ng/mL, greater than about 0.95 ng/mL,greater than about 1.0 ng/mL, greater than about 1.1 ng/mL, greater thanabout 1.2 ng/mL, greater than about 1.3 ng/mL, greater than about 1.4ng/mL, greater than about 1.5 ng/mL, greater than about 1.6 ng/mL,greater than about 1.7 ng/mL, greater than about 1.8 ng/mL, greater thanabout 1.9 ng/mL, greater than about 2.0 ng/mL, greater than about 2.1ng/mL, greater than about 2.2 ng/mL, greater than about 2.3 ng/mL,greater than about 2.4 ng/mL, greater than about 2.5 ng/mL, greater thanabout 2.75 ng/mL, or greater than about 3.0 ng/mL.

Particular embodiments of the present disclosure comprise a mean IL-10serum trough concentration in a range of from about 0.1 ng/mL to about1.0 ng/mL, from about 0.1 ng/ml to about 0.9 ng/mL, from about 0.1ng/mL, to about 0.8 ng/ml, from about 0.1 ng/mL to about 0.7 ng/mL, fromabout 0.1 ng/mL to about 0.6 ng/mL, from about of 0.1 ng/mL to about 0.5ng/mL, from about 0.2 ng/mL to about 1.0 ng/mL, from about 0.2 ng/mL toabout 0.9 ng/mL, from about 0.2 ng/mL to about 0.8 ng/mL, from about 0.2ng/mL to about 0.7 ng/mL, from about 0.2 ng/mL to about 0.6 ng/mL, fromabout 0.2 ng/mL to about 0.5 ng/mL, from about 0.3 ng/mL to about 1.0ng/mL, from about 0.3 ng/mL to about 0.9 ng/mL, from about 0.3 ng/mL toabout 0.8 ng/mL, from about 0.3 ng/mL to about 0.7 ng/mL, from about 0.3ng/mL to about 0.6 ng/mL, from about 0.3 ng/mL to about 0.5 ng/mL, fromabout 0.3 ng/mL to about 0.4 ng/mL, from about 0.4 ng/mL to about 1.0ng/mL, from about 0.4 ng/mL to about 0.9 ng/mL, from about 0.4 ng/mL toabout 0.8 ng/mL, from about 0.4 ng/mL to about 0.7 ng/mL, from about 0.4ng/mL to about 0.6 ng/mL, from about 0.4 ng/mL to about 0.5 ng/mL, fromabout 0.5 ng/mL to about 1.0 ng/mL, from about 0.5 ng/mL to about 0.9ng/mL, from about 0.5 ng/mL to about 0.8 ng/mL, from about 0.5 ng/mL toabout 0.7 ng/mL, from about 0.5 ng/mL to about 0.6 ng/mL, from about 0.7ng/mL to about 2.3 n/mL, from about 0.8 ng/mL to about 2.2 ng/mL, fromabout 0.9 ng/mL to about 2.1 ng/mL, from about 1.0 ng/mL to about 2.1ng/mL, from about 1.0 ng/mL to about 2.0 ng/mL, from about 1.0 ng/mL toabout 1.9 ng/mL, from about 1.0 ng/mL to about 1.8 ng/mL, from about 1.0ng/mL to about 1.7 ng/mL, from about 1.0 ng/mL to about 1.6 ng/mL, fromabout 1.0 ng/mL to about 1.5 ng/mL, from about 1.9 ng/mL to greater thanabout 2.5 ng/mL, from about 1.9 ng/mL to about 2.5 ng/mL, from about 1.9ng/mL to about 2.4 ng/mL, from about 1.9 ng/mL to about 2.3 ng/mL, fromabout 1.9 ng/mL to about 2.2 ng/mL, or from about 1.9 ng/mL to about 2.1ng/mL.

In particular embodiments directed to the treatment or prevention ofanti-inflammatory diseases, disorders or conditions, therapy isoptimized by achieving a mean IL-10 serum trough concentration of 0.1ng/mL to 1.0 ng/mL, of 0.1 ng/mL to 0.9 ng/mL, of 0.1 ng/mL to 0.8ng/mL, of 0.1 ng/mL to 0.7 ng/mL, of 0.1 ng/mL to 0.6 ng/mL, of 0.1ng/mL to 0.5 ng/mL, of 0.2 ng/mL to 1.0 ng/mL, of 0.2 ng/mL to 0.9ng/mL, of 0.2 ng/mL to 0.8 ng/mL, of 0.2 ng/mL to 0.7 ng/mL, of 0.2ng/mL to 0.6 ng/mL, of 0.2 ng/mL to 0.5 ng/mL, of 0.3 ng/mL to 1.0ng/mL, of 0.3 ng/mL to 0.9 ng/mL, of 0.3 ng/mL to 0.8 ng/mL, of 0.3ng/mL to 0.7 ng/mL, of 0.3 ng/mL to 0.6 ng/mL, of 0.3 ng/mL to 0.5ng/mL, of 0.3 ng/mL to 0.4 ng/mL, of 0.4 ng/mL to 1.0 ng/mL, of 0.4ng/mL to 0.9 ng/mL, of 0.4 ng/mL to 0.8 ng/mL, of 0.4 ng/mL to 0.7ng/mL, of 0.4 ng/mL to 0.6 ng/mL, of 0.4 ng/mL to 0.5 ng/mL, of 0.5ng/mL to 1.0 ng/mL, of 0.5 ng/mL to 0.9 ng/mL, of 0.5 ng/mL to 0.8ng/mL, of 0.5 ng/mL to 0.7 ng/mL, or of 0.5 ng/mL to 0.6 ng/mL.

The Experimental section describes evaluations of the therapeuticefficacy of mIL-10 and PEG-mIL-10 in PDV6 squamous cell carcinoma andCT-26 colon carcinoma, wherein the mIL-10 and mPEG-IL-10 dosingparameters (amount and frequency of administration) are sufficient toachieve a mean IL-10 serum trough concentration of 1-2 ng/mL. Asdescribed in the Experimental section, PEG-IL-10 treatment resulted in acomplete response, whereas IL-10 treatment demonstrated anti-tumorfunction but not a complete response.

The effect of IL-10 treatment in hepatitis C can also be evaluated. Amouse model with a functional immune system that is susceptible toinfection by the hepatitis C virus (see Dorner, M. (9 Jun. 2011) Nature474:208-211) can be utilized to evaluate the pharmacokinetic andpharmacodynamic effects of mIL-10 and PEG-mIL-10. Using the teachingsset forth herein and the knowledge base of the skilled artisan, theeffect of mIL-10 and PEG-mIL-10 administered to achieve a mean IL-10serum trough concentration of about 0.1 ng/mL, about 0.5 ng/mL, about1.0 ng/mL, about 1.5 ng/mL and about 2 ng/mL can be assessed.

Although not prevalent at therapeutic doses in most patient populations,administration of higher doses of IL-10 has caused adverse effects(e.g., headache, anemia and effects on the liver) in a limited number ofsubjects. Fortunately, such adverse effects are not prevalent when amean IL-10 serum concentration of 0.1-2 ng/mL is maintained over theduration of treatment. Nonetheless, another embodiment of the presentdisclosure provides a method for monitoring a subject receiving IL-10therapy to predict, and thus potentially avoid, adverse effects, themethod comprising: (1) measuring the subject's peak concentration ofIL-10; (2) measuring the subject's trough concentration of IL-10; (3)calculating a peak-trough fluctuation; and, (4) using the calculatedpeak-trough fluctuation to predict potential adverse effects in thesubject. A smaller peak-trough fluctuation indicates a lower probabilitythat the subject will experience IL-10-related adverse effects. Incertain embodiments, particular peak-trough fluctuations are determinedfor the treatment of particular diseases, disorders and conditions usingparticular dosing parameters, and those fluctuations are used asreference standards.

In addition to the IL-10 dosing-related parameters described above,volume of distribution considerations are also pertinent. For themajority of drugs, plasma drug concentrations decline in amulti-exponential fashion. Immediately after intravenous administration,the drug rapidly distributes throughout an initial space (minimallydefined as the plasma volume), and then a slower, equilibrativedistribution to extravascular spaces (e.g., certain tissues) occurs.Intravenous IL-10 administration is associated with such atwo-compartment kinetic model (see Rachmawati, H, et al. (2004) Pharm.Res. 21(11):2072-78). The pharmacokinetics of subcutaneous recombinanthIL-10 has also been studied (Radwanski, E, et al. (1998) Pharm. Res.15(12):1895-1901). Moreover, IL-10 modifications have been introduced inan attempt to target the cytokine to specific cell types (seeRachmawati, H. (May 2007) Drug Met. Dist. 35(5):814-21).

As described further hereafter, the IL-10 and PEG-IL-10 anti-tumorefficacy observed in mice results from induction of cytotoxic enzymes inCD8+ T cells, resulting in the killing of tumor cells. Many anti-cancercompounds, including, but not limited to, apoptosis-inducing agents, areadministrated in cycles. Frequently, a single dose or a series of dosesapproaching the Maximum Tolerated Dose (MTD) are administered, includinga single application or series of high doses approaching the maximallytolerated dose (MTD), followed by a cessation of dosing (a “drugholiday”) to allow recovery of the patient's normal physiology. By wayof example, this dosing strategy is applied to cytotoxicchemotherapeutic antibody therapies, such as anti-VEGF (AVASTIN), and toshort-lived biologic reagents, such as PROLEUKIN (IL-2).

Murine studies were performed to generate data helpful in understandingthe pharmacokinetic parameters of IL-10 therapy and in optimizing thetumor treatment regimens in humans. As described in the Experimentalsection, although mice receiving the same amount of drug over the courseof a week administered in either one or several doses had similaroverall exposures, mice receiving daily doses exhibited the greatestreduction in tumor size (Table 15). Moreover, treatment regimens thatresulted in maintenance of serum trough concentrations greater thanabout 1 ng/mL (e.g., 1.1-2.1 ng/mL) exhibited the greatest reduction intumor size and weight (Table 16).

The present disclosure contemplates administration of any dose thatresults in maintenance of serum trough concentrations greater than about0.1 ng/mL (e.g., 0.1-2 ng/mL, 0.1-1 ng/mL, 0.5-1.5 ng/mL or 1.1-2.1ng/mL). For example, when the subject is a human, non-pegylated hIL-10may be administered at a dose greater than 15 μg/kg/day, greater than 18μg/kg/day, greater than 20 μg/kg/day, greater than 21 μg/kg/day, greaterthan 22 μg/kg/day, greater than 23 μg/kg/day, greater than 24 μg/kg/day,or greater than 25 μg/kg/day. When the subject is a human, PEG-hIL-10comprising a relatively small PEG (e.g., 5 kDa mono-di PEG-hIL0) may beadministered at a dose greater than 2.0 μg/kg/day, greater than 2.3μg/kg/day, greater than 2.5 μg/kg/day, greater than 2.6 μg/kg/day,greater than 2.7 μg/kg/day, greater than 2.8 μg/kg/day, greater than 2.9μg/kg/day, greater than 3.0 μg/kg/day, greater than 3.1 μg/kg/day,greater than 3.2 μg/kg/day, greater than 3.3 μg/kg/day, greater than 3.4μg/kg/day or greater than 3.5 μg/kg/day.

The Role of CD8+ T Cells in IL-10 Function

CD8 (cluster of differentiation 8) is a transmembrane glycoprotein thatserves as a co-receptor for the T cell receptor (TCR). The CD8co-receptor is predominantly expressed on the surface of cytotoxic Tlymphocytes (CTL), but it is also found on other cell types, includingnatural killer cells (NK). Like the TCR, CD8 binds to a majorhistocompatibility complex (MHC) molecule, but is specific for the ClassI MHC protein.

CD8 function requires formation of a dimer comprising a pair of CD8chains. There are two isoforms of CD8, alpha and beta, and the mostcommon form of CD8 comprises a CD8-α and a CD8-β chain, both members ofthe immunoglobulin superfamily. CD8-α interacts with the Class I MHCmolecule, and this interaction keeps the T cell receptor of thecytotoxic T cell and the target cell closely bound duringantigen-specific activation. Cytotoxic T cells with CD8 surface proteinare referred to as “CD8+ T cells”. CD8+ T cells (CTL and NK cells)recognize antigens (generally cell-surface peptides or proteinsresulting from infection by intracellular pathogens) of specificinfected target cells, and if those antigens differ from the normalantigen profile of the subject (“immunologic self”), the CD8+ T cellsbecome activated and induce apoptosis of the target cells.

Several scenarios exist wherein antigen profiles differ. For example,when a pathogen (e.g., a virus) invades a cell, the cell produces“non-self” cell surface antigens, and CD8|T cells initiate animmunological response in an attempt to eradicate the infected cells.Another scenario occurs wherein some of a cell's proteins are modifieddue to mutations at the nucleic acid and/or the amino acid level. Cancercells generally carry many mutations and are recognized as ‘different’by CD8+ T cells. The presence of CD8+ T cells in human cancer correlateswith longer survival.

In both of the aforementioned scenarios, activated CD8+ T cells produceIFNγ, perforin and Granzyme B. IFNγ is important to further up-regulatethe “presentation” of antigens on the target cells, which occurs onClass I MHC protein. Perforin and Granzyme B mediate the killing of thetarget cell (e.g., virus and cancer).

Perforin, a cytolytic protein found in the granules of CTLs and NKs,inserts itself into a target cell's plasma membrane upon degranulation.Perforin has structural and functional similarities to complementcomponent 9 (C9), and, like C9, perforin creates transmembrane tubulesand is capable of non-specifically lysing a variety of target cells.Perforin is a key effector molecule for T-cell- and NK-cell-mediatedcytolysis.

As alluded to above, Granzyme B is a serine protease expressed bycytotoxic T lymphocytes (CTL) and natural killer (NK) cells. CTL and NKcells recognize specific infected target cell populations and induceapoptosis of cells that bear on their surface ‘non-self’ antigens,usually peptides or proteins resulting from infection by intracellularpathogens. Granzyme B is crucial for the rapid induction of target cellapoptosis by CTL in cell-mediated immune response.

IL-10 plays diverse roles in the activation of CD8+ T cells. Forexample, IL-10 induces the effector molecules (IFNγ, perforin andGranzyme B) in memory CD8+ T cells, cells which have been generatedduring a previous infection or vaccination. Such memory CD8+ T cells arethe cells responsible for providing a subject's long-term protectionagainst viruses. Although generation and amplification of memory CD8+ Tcells may occur when IL-10 is not present (Vicari, A, and Trinchieri, G.(2004) Immuno. Rev. 202:223-236), the fact that IL-10 directly activatessuch cells provides a unique and alternative therapeutic approach.Though chronic viral infection has been linked to CD8+ T cells (Virgin,H, et al. (2009) Cell 138, p. 30), treatment of subjects (e.g., mice)with unpegylated IL-10 or pegylated IL-10 has not been described.

In view of the above, an embodiment of the present disclosure is basedon the nexus between CD8+ T cells and both cancer and viral infections.Thus, certain methods of treating and/or preventing cancer-relateddiseases, disorders and conditions, such as maintaining a mean IL-10serum concentration of, e.g., ≥0.5 ng/mL, ≥1 ng/mL, or ≥2 ng/mL, shouldalso be applicable in the treatment of viral-related diseases, disordersand conditions.

In contrast to other cytokines, IL-10 can be deemed both a potentimmunostimulatory and immunosuppressive factor. The role of CD8+ T cellsin chronic inflammation has not been completely elucidated. However,because the involvement of IFNγ in cancer and viral-related disorders ismediated, at least in part, through CD8+ T cells, and because theIL-10-T cell pathway implicated in the control of inflammation-relateddisorders (through down-regulation of inflammatory cytokines) alsoinvolves IFNγ, CD8+ T cells may also play a key role in inflammation.Thus, IL-10 may prove to be an important therapeutic in the currentstable of anti-inflammatory agents.

Methods of Production of IL-10

A polypeptide of the present disclosure can be produced by any suitablemethod, including non-recombinant (e.g., chemical synthesis) andrecombinant methods.

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as9-fluorenylmethoxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc), areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical syntheses are known in the art (e.g., Ganesan A.(2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J. A, et al., (2005)Protein Pept Lett. 12:723-8).

Solid phase peptide synthesis may be performed as described hereafter.The alpha functions (Nα) and any reactive side chains are protected withacid-labile or base-labile groups. The protective groups are stableunder the conditions for linking amide bonds but can readily be cleavedwithout impairing the peptide chain that has formed. Suitable protectivegroups for the α-amino function include, but are not limited to, thefollowing: Boc, benzyloxycarbonyl (Z). O-chlorbenzyloxycarbonyl,bi-phenylisopropyloxycarbonyl, tert-amyloxycarbonyl (Amoc), α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, Fmoc,1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.

Suitable side chain protective groups include, but are not limited to:acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl),benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom),o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis, the C-terminal amino acid is coupled to asuitable support material. Suitable support materials are those whichare inert towards the reagents and reaction conditions for the step-wisecondensation and cleavage reactions of the synthesis process and whichdo not dissolve in the reaction media being used. Examples ofcommercially-available support materials include styrene/divinylbenzenecopolymers which have been modified with reactive groups and/orpolyethylene glycol; chloromethylated styrenedivinylbenzene copolymers;hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers;and the like. When preparation of the peptidic acid is desired,polystyrene (1%)-divinylbenzene or TentaGel®) derivatized with4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloride canbe used. In the case of the peptide amide, polystyrene (1%)divinylbenzene or TentaGel® derivatized with5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid (PAL-anchor) orp-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rink amide anchor)can be used.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material by theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran.N-methylpyrolidone or similar solvents at room temperature or elevatedtemperatures (e.g., between 40° C. and 60° C.) and with reaction timesof, e.g., 2 to 72 hours.

The coupling of the Nα-protected amino acid (e.g., the Fmoc amino acid)to the PAL, Wang or Rink anchor can, for example, be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, O-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, e.g., with theaid of TBTU with addition of HOBt, with or without the addition of abase such as, for example, diisopropylethylamine (DIEA), triethylamineor N-methylmorpholine, e.g., diisopropylethylamine with reaction timesof 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the aminoacid and the coupling reagents, for example, in a 2-fold excess and attemperatures between about 10° C. and 50° C., for example, 25° C., in asolvent such as dimethylformamide, N-methylpyrrolidone ordichloromethane, e.g., dimethylformamide).

Instead of the coupling reagents, it is also possible to use the activeesters (e.g., pentafluorophenyl, p-nitrophenyl or the like), thesymmetric anhydride of the Nα-Fmoc-amino acid, its acid chloride or acidfluoride, under the conditions described above.

The Nα-protected amino acid (e.g., the Fmoc amino acid) can be coupledto the 2-chlorotrityl resin in dichloromethane with the addition of DIEAand having reaction times of 10 to 120 minutes, e.g., 20 minutes, but isnot limited to the use of this solvent and this base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis, typically in anautomated peptide synthesizer. After cleavage of the Nα-Fmoc protectivegroup of the coupled amino acid on the solid phase by treatment with,e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes,e.g., 2×2 minutes with 50% piperidine in DMF and 1×15 minutes with 20%piperidine in DMF, the next protected amino acid in a 3 to 10-foldexcess, e.g., in a 10-fold excess, is coupled to the previous amino acidin an inert, non-aqueous, polar solvent such as dichloromethane, DMF ormixtures of the two and at temperatures between about 10° C. and 50° C.,e.g., at 25° C. The previously mentioned reagents for coupling the firstNα-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V of scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, e.g., 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1, within 0.5 to 3 hours,e.g., 2 hours. Peptides with fully protected side chains are obtained bycleaving the 2-chlorotrityl anchor with glacial aceticaciditrifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C. (e.g.,about 25° C.), and reaction times between about 12 and 24 hours (e.g.,about 18 hours). In addition, the peptide can be cleaved from thesupport by re-esterification, e.g., with methanol.

The acidic solution that is obtained may be admixed with a 3 to 20-foldamount of cold ether or n-hexane, e.g., a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-precipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedcan be taken up in water or tert-butanol or mixtures of the twosolvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods,including ion exchange over a weakly basic resin in the acetate form;hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography, e.g., oncarboxymethyl cellulose; distribution chromatography, e.g., on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) e.g., reversed-phase HPLC on octyl oroctadecylsilylsilica (ODS) phases.

B. Recombinant Production

Methods describing the preparation of human and mouse IL-10 can be foundin, for example, U.S. Pat. No. 5,231,012, which teaches methods for theproduction of proteins having IL-10 activity, including recombinant andother synthetic techniques. IL-10 can be of viral origin, and thecloning and expression of a viral IL-10 from Epstein Barr virus (BCRF1protein) is disclosed in Moore et al., (1990) Science 248:1230. IL-10can be obtained in a number of ways using standard techniques known inthe art, such as those described herein. Recombinant human IL-10 is alsocommercially available, e.g., from PeproTech, Inc., Rocky Hill, N.J.

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that may be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they mayinclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1); and hamster cells (e.g., Chinese hamster ovary (CIIO)cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification. In oneembodiment, the protein may be isolated using metal chelatechromatography methods. Proteins may contain modifications to facilitateisolation.

The polypeptides may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide may be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than about 90%, less thanabout 60%, less than about 50%, less than about 40%, less than about30%, less than about 20%, less than about 10%, less than about 5%, orless than about 1%.

An IL-10 polypeptide may be generated using recombinant techniques tomanipulate different IL-10-related nucleic acids known in the art toprovide constructs capable of encoding the IL-10 polypeptide. It will beappreciated that, when provided a particular amino acid sequence, theordinary skilled artisan will recognize a variety of different nucleicacid molecules encoding such amino acid sequence in view of herbackground and experience in, for example, molecular biology.

Amide Bond Substitutions

In some cases, IL-10 includes one or more linkages other than peptidebonds, e.g., at least two adjacent amino acids are joined via a linkageother than an amide bond. For example, in order to reduce or eliminateundesired proteolysis or other means of degradation, and/or to increaseserum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of IL-10 can besubstituted.

In another example, one or more amide linkages (—CO—NH—) in IL-10 can bereplaced with a linkage which is an isostere of an amide linkage, suchas —CH₂NH—, —CH₂S—, —CH₂CH₂—, —CH═CH-(cis and trans), —COCH₂—,—CH(OH)CH₂— or —CH₂SO—. One or more amide linkages in IL-10 can also bereplaced by, for example, a reduced isostere pseudopeptide bond. SeeCouder et al. (1993) Int. J. Peptide Protein Res. 41:181-184. Suchreplacements and how to effect them are known to those of ordinary skillin the art.

Amino Acid Substitutions

One or more amino acid substitutions can be made in an IL-10polypeptide. The following are non-limiting examples:

a) substitution of alkyl-substituted hydrophobic amino acids, includingalanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyricacid, (S)-cyclohexylalanine or other simple alpha-amino acidssubstituted by an aliphatic side chain from C₁-C₁₀ carbons includingbranched, cyclic and straight chain alkyl, alkenyl or alkynylsubstitutions;

b) substitution of aromatic-substituted hydrophobic amino acids,including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-4-tetrahydropyridyl)-alanine,N,N-gamma, gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination), carboxylic acids or any of the manywell-known activated derivatives such as acid chlorides, active esters,active azolides and related derivatives, and lysine, omithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids.

e) substitution of side chain amide residues, including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine; and

f) substitution of hydroxyl-containing amino acids, including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some cases, IL-10 comprises one or more naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids, orD-enantiomers of an amino acid. For example, IL-10 can comprise onlyD-amino acids. For example, an IL-10 polypeptide can comprise one ormore of the following residues: hydroxyproline, β-alanine,o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid,m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyricacid, N-methylglycine (sarcosine), omithine, citrulline, t-butylalanine,t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine,norleucine, naphthylalanine, pyridylalanine 3-benzothienyl alanine,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine,4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine,methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyricacid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine,ε-amino hexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid,ω-aminooctanoic acid, σ-aminodecanoic acid, ω-aminotetradecanoic acid,cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid,δ-amino valeric acid, and 2,3-diaminobutyric acid.

Additional Modifications

A cysteine residue or a cysteine analog can be introduced into an IL-10polypeptide to provide for linkage to another peptide via a disulfidelinkage or to provide for cyclization of the IL-10 polypeptide. Methodsof introducing a cysteine or cysteine analog are known in the art; see,e.g., U.S. Pat. No. 8,067,532.

An IL-10 polypeptide can be cyclized. One or more cysteines or cysteineanalogs can be introduced into an IL-10 polypeptide, where theintroduced cysteine or cysteine analog can form a disulfide bond with asecond introduced cysteine or cysteine analog. Other means ofcyclization include introduction of an oxime linker or a lanthioninelinker, see, e.g., U.S. Pat. No. 8,044,175. Any combination of aminoacids (or non-amino acid moieties) that can form a cyclizing bond can beused and/or introduced. A cyclizing bond can be generated with anycombination of amino acids (or with an amino acid and —(CH₂)_(n)—CO— or—(CH₂)_(n)—C₆H₄—CO—) with functional groups which allow for theintroduction of a bridge. Some examples are disulfides, disulfidemimetics such as the —(CH2)_(n)— carba bridge, thioacetal, thioetherbridges (cystathionine or lanthionine) and bridges containing esters andethers. In these examples, n can be any integer, but is frequently lessthan ten.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives include C-terminalhydroxymethyl derivatives, o-modified derivatives (e.g., C-terminalhydroxymethyl benzyl ether), N-terminally modified derivatives includingsubstituted amides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in an IL-10 polypeptide isreplaced with one or more D-amino acids.

In some cases, an IL-10 polypeptide is a retroinverso analog (see, e.g.,Sela and Zisman (1997) FASEB J. 11:449). Retro-inverso peptide analogsare isomers of linear polypeptides in which the direction of the aminoacid sequence is reversed (retro) and the chirality, D- or L-, of one ormore amino acids therein is inverted (inverso), e.g., using D-aminoacids rather than L-amino acids. [See, e.g., Jameson et al. (1994)Nature 368:744; and Brady et al. (1994) Nature 368:692].

An IL-10 polypeptide can include a “Protein Transduction Domain” (PTD),which refers to a polypeptide, polynucleotide, carbohydrate, or organicor inorganic molecule that facilitates traversing a lipid bilayer,micelle, cell membrane, organelle membrane, or vesicle membrane. A PTDattached to another molecule facilitates the molecule traversing amembrane, for example going from extracellular space to intracellularspace, or cytosol to within an organelle. In some embodiments, a PTD iscovalently linked to the amino terminus of an IL-10 polypeptide, whilein other embodiments, a PTD is covalently linked to the carboxylterminus of an IL-10 polypeptide. Exemplary protein transduction domainsinclude, but are not limited to, a minimal undecapeptide proteintransduction domain (corresponding to residues 47-57 of HIV-1 TATcomprising YGRKKRRQRRR; SEQ ID NO://); a polyarginine sequencecomprising a number of arginine residues sufficient to direct entry intoa cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); aDrosophila Antennapedia protein transduction domain (Noguchi et al.(2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide(Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wenderet al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR(SEQ ID NO:/); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:/);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:/); and RQIKIWFQNRRMKWKK(SEQ ID NO:/). Exemplary PTDs include, but are not limited to,YGRKKRRQRRR (SEQ ID NO:/), RKKRRQRRR (SEQ ID NO:/); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following:

(SEQ ID NO: //) YGRKKRRQRRR; (SEQ ID NO: //) RKKRRQRR; (SEQ ID NO: //)YARAAARQARA; (SEQ ID NO: //) THRLPRRRRRR; and (SEQ ID NO: //)GGRRARRRRRR.

The carboxyl group COR₃ of the amino acid at the C-terminal end of anIL-10 polypeptide can be present in a free form (R₃═OH) or in the formof a physiologically-tolerated alkaline or alkaline earth salt such as,e.g., a sodium, potassium or calcium salt. The carboxyl group can alsobe esterified with primary, secondary or tertiary alcohols such as,e.g., methanol, branched or unbranched C₁-C₆-alkyl alcohols, e.g., ethylalcohol or tert-butanol. The carboxyl group can also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆-alkylamines or C₁-C₆ di-alkylamines, e.g., methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminus of an IL-10polypeptide can be present in a free form (R₁═H and R₂═H) or in the formof a physiologically-tolerated salt such as, e.g., a chloride oracetate. The amino group can also be acetylated with acids such thatR₁═H and R₂=acetyl, trifluoroacetyl, or adamantyl. The amino group canbe present in a form protected by amino-protecting groups conventionallyused in peptide chemistry, such as those provided above (e.g., Fmoc,Benzyloxy-carbonyl (Z), Boc, and Alloc). The amino group can beN-alkylated in which R₁ and/or R₂═C₁-C₆ alkyl or C₂-C₅ alkenyl or C₁-C₉aralkyl. Alkyl residues can be straight-chained, branched or cyclic(e.g., ethyl, isopropyl and cyclohexyl, respectively).

Particular Modifications to Enhance and/or Mimic IL-10 Function

It is frequently beneficial, and sometimes imperative, to improve one ofmore physical properties of the treatment modalities disclosed herein(e.g., IL-10) and/or the manner in which they are administered.Improvements of physical properties include, for example, modulatingimmunogenicity; methods of increasing water solubility, bioavailability,serum half-life, and/or therapeutic half-life; and/or modulatingbiological activity. Certain modifications may also be useful to, forexample, raise of antibodies for use in detection assays (e.g., epitopetags) and to provide for ease of protein purification. Such improvementsmust generally be imparted without adversely impacting the bioactivityof the treatment modality and/or increasing its immunogenicity.

Pegylation of IL-10 is one particular modification contemplated by thepresent disclosure, while other modifications include, but are notlimited to, glycosylation (N- and O-linked); polysialylation; albuminfusion molecules comprising serum albumin (e.g., human serum albumin(HSA), cyno serum albumin, or bovine serum albumin (BSA)); albuminbinding through, for example a conjugated fatty acid chain (acylation);and Fc-fusion proteins.

Pegylation:

The clinical effectiveness of protein therapeutics is often limited byshort plasma half-life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by various modifications,including conjugating or linking the polypeptide sequence to any of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes. This is frequently effectedby a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG. Such PEG-conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity.

In addition to the beneficial effects of pegylation on pharmacokineticparameters, pegylation itself may enhance activity. For example,PEG-IL-10 has been shown to be more efficacious against certain cancersthan unpegylated IL-10 (see, e.g., EP 206636A2).

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. A molecular weight of the PEGused in the present disclosure is not restricted to any particularrange, and examples are set forth elsewhere herein; by way of example,certain embodiments have molecular weights between 5 kDa and 20 kDa,while other embodiments have molecular weights between 4 kDa and 10 kDa.

The present disclosure also contemplates compositions of conjugateswherein the PEGs have different n values, and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. Exemplary reactionconditions are described throughout the specification. Cation exchangechromatography may be used to separate conjugates, and a fraction isthen identified which contains the conjugate having, for example, thedesired number of PEGs attached, purified free from unmodified proteinsequences and from conjugates having other numbers of PEGs attached.

Pegylation most frequently occurs at the alpha amino group at theN-terminus of the polypeptide, the epsilon amino group on the side chainof lysine residues, and the imidazole group on the side chain ofhistidine residues. Since most recombinant polypeptides possess a singlealpha and a number of epsilon amino and imidazole groups, numerouspositional isomers can be generated depending on the linker chemistry.General pegylation strategies known in the art can be applied herein.PEG may be bound to a polypeptide of the present disclosure via aterminal reactive group (a “spacer”) which mediates a bond between thefree amino or carboxyl groups of one or more of the polypeptidesequences and polyethylene glycol. The PEG having the spacer which maybe bound to the free amino group includes N-hydroxysuccinylimidepolyethylene glycol which may be prepared by activating succinic acidester of polyethylene glycol with N-hydroxysuccinylimide. Anotheractivated polyethylene glycol which may be bound to a free amino groupis 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which maybe prepared by reacting polyethylene glycol monomethyl ether withcyanuric chloride. The activated polyethylene glycol which is bound tothe free carboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the presentdisclosure to PEG having a spacer may be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to protein of from 4:1 to 30:1. Reaction conditions maybe selected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH≥7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art may be used to terminate thereaction. In some embodiments the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C. Pegylation ofvarious molecules is discussed in, for example, U.S. Pat. Nos.5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263. PEG-IL-10 isdescribed in, e.g., U.S. Pat. No. 7,052,686. Specific reactionconditions contemplated for use herein are set forth in the Experimentalsection.

The present disclosure also contemplates the use of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix′ XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation:

For purposes of the present disclosure, “glycosylation” is meant tobroadly refer to the enzymatic process that attaches glycans toproteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Glycosylation can dramatically affect the physical properties (e.g.,solubility) of polypeptides such as IL-10 and can also be important inprotein stability, secretion, and subcellular localization. Glycosylatedpolypeptides may also exhibit enhanced stability or may improve one ormore pharmacokinetic properties, such as half-life. In addition,solubility improvements can, for example, enable the generation offormulations more suitable for pharmaceutical administration thanformulations comprising the non-glycosylated polypeptide.

Proper glycosylation can be essential for biological activity. In fact,some genes from eukaryotic organisms, when expressed in bacteria (e.g.,E. coli) which lack cellular processes for glycosylating proteins, yieldproteins that are recovered with little or no activity by virtue oftheir lack of glycosylation.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment of thepresent disclosure comprises the generation and use of N-glycosylationvariants.

The polypeptide sequences of the present disclosure may optionally bealtered through changes at the nucleic acid level, particularly bymutating the nucleic acid encoding the polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids. Another means of increasing the number of carbohydratemoieties on the polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Removal of carbohydrates may beaccomplished chemically or enzymatically, or by substitution of codonsencoding amino acid residues that are glycosylated. Chemicaldeglycosylation techniques are known, and enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases.

Dihydrofolate reductase (DHFR)-deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins. These cells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2.6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

Polysialylation:

The present disclosure also contemplates the use of polysialylation, theconjugation of polypeptides to the naturally occurring, biodegradableα-(2→8) linked polysialic acid (“PSA”) in order to improve thepolypeptides' stability and in vivo pharmacokinetics. PSA is abiodegradable, non-toxic natural polymer that is highly hydrophilic,giving it a high apparent molecular weight in the blood which increasesits serum half-life. In addition, polysialylation of a range of peptideand protein therapeutics has led to markedly reduced proteolysis,retention of in vivo activity, and reduction in immunogenicity andantigenicity (see, e.g., G. Gregoriadis et al., Int. J. Pharmaceutics300(1-2):125-30). As with modifications with other conjugates (e.g.,PEG), various techniques for site-specific polysialylation are available(see, e.g., T. Lindhout et al., (2011) PNAS 108(18)7397-7402).

Albumin Fusion:

Additional suitable components and molecules for conjugation includealbumins such as human serum albumin (HSA), cyno serum albumin, andbovine serum albumin (BSA).

Mature HSA, a 585 amino acid polypeptide (˜67 kDa) having a serumhalf-life of ˜20 days, is primarily responsible for the maintenance ofcolloidal osmotic blood pressure, blood pH, and transport anddistribution of numerous endogenous and exogenous ligands. The proteinhas three structurally homologous domains (domains I, II and Ill), isalmost entirely in the alpha-helical conformation, and is highlystabilized by 17 disulphide bridges. The three primary drug bindingregions of albumin are located on each of the three domains withinsub-domains IB, IIA and IIIA.

Albumin synthesis takes place in the liver, which produces theshort-lived, primary product preproalbumin. Thus, the full-length HSAhas a signal peptide of 18 amino acids (MKWVTFISLLFLFSSAYS; SEQ IDNO://) followed by a pro-domain of 6 amino acids (RGVFRR; SEQ ID NO:/1);this 24 amino acid residue peptide may be referred to as the pre-prodomain. HSA can be expressed and secreted using its endogenous signalpeptide as a pre-pro-domain. Alternatively, HSA can be expressed andsecreted using a IgK signal peptide fused to a mature construct.Preproalbumin is rapidly co-translationally cleaved in the endoplasmicreticulum lumen at its amino terminus to produce the stable, 609-aminoacid precursor polypeptide, proalbumin. Proalbumin then passes to theGolgi apparatus, where it is converted to the 585 amino acid maturealbumin by a furin-dependent amino-terminal cleavage.

The primary amino acid sequences, structure, and function of albuminsare highly conserved across species, as are the processes of albuminsynthesis and secretion. Albumin serum proteins comparable to HSA arefound in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats.Of the non-human species, bovine serum albumin (BSA) is the moststructurally similar to HSA (see, e.g., Kosa et al., November 2007 JPharm Sci. 96(11):3117-24). The present disclosure contemplates the useof albumin from non-human species, including, but not limited to, thoseset forth above, in, for example, the drug development process.

According to the present disclosure, albumin may be conjugated to a drugmolecule (e.g., a polypeptide described herein) at the carboxylterminus, the amino terminus, both the carboxyl and amino termini, andinternally (see, e.g., U.S. Pat. Nos. 5,876,969 and 7,056,701).

In the HSA-drug molecule conjugates contemplated by the presentdisclosure, various forms of albumin may be used, such as albuminsecretion pre-sequences and variants thereof, fragments and variantsthereof, and HSA variants. Such forms generally possess one or moredesired albumin activities. In additional embodiments, the presentdisclosure involves fusion proteins comprising a polypeptide drugmolecule fused directly or indirectly to albumin, an albumin fragment,and albumin variant, etc., wherein the fusion protein has a higherplasma stability than the unfused drug molecule and/or the fusionprotein retains the therapeutic activity of the unfused drug molecule.In some embodiments, the indirect fusion is effected by a linker, suchas a peptide linker or modified version thereof.

Intracellular cleavage may be carried out enzymatically by, for example,furin or caspase. Cells express a low level of these endogenous enzymes,which are capable of cleaving a portion of the fission moleculesintracellularly; thus, some of the polypeptides are secreted from thecell without being conjugated to HSA, while some of the polypeptides aresecreted in the form of fusion molecules that comprise HSA. Embodimentsof the present disclosure contemplate the use of various furin fusionconstructs. For example, constructs may be designed that comprise thesequence RGRR, RKRKKR, RKKR, or RRRKKR.

The present disclosure also contemplates extra-cellular cleavage (i.e.,ex-vivo cleavage) whereby the fusion molecules are secreted from thecell, subjected to purification, and then cleaved. It is understood thatthe excision may dissociate the entire HSA-linker complex from themature IL-10, or less that the entire HSA-linker complex.

As alluded to above, fusion of albumin to one or more polypeptides ofthe present disclosure can, for example, be achieved by geneticmanipulation, such that the nucleic acid coding for HSA, or a fragmentthereof, is joined to the nucleic acid coding for the one or morepolypeptide sequences. Thereafter, a suitable host can be transformed ortransfected with the fused nucleotide sequences in the form of, forexample, a suitable plasmid, so as to express a fusion polypeptide. Theexpression may be effected in vitro from, for example, prokaryotic oreukaryotic cells, or in vivo from, for example, a transgenic organism.In some embodiments of the present disclosure, the expression of thefusion protein is performed in mammalian cell lines, for example, CHOcell lines. Transformation is used broadly herein to refer to thegenetic alteration of a cell resulting from the direct uptake throughthe cell membrane, incorporation and expression of exogenous geneticmaterial (exogenous nucleic acid). Transformation occurs naturally insome species of bacteria, but it can also be effected by artificialmeans in other cells.

Furthermore, albumin itself may be modified to extend its circulatinghalf-life. Fusion of the modified albumin to IL-10 can be attained bythe genetic manipulation techniques described above or by chemicalconjugation; the resulting fusion molecule has a half-life that exceedsthat of fusions with non-modified albumin (see WO2011/051489).

Alternative Albumin Binding Strategies:

Several albumin-binding strategies have been developed as alternativesto direct fusion, including albumin binding through a conjugated fattyacid chain (acylation). Because serum albumin is a transport protein forfatty acids, these natural ligands with albumin-binding activity havebeen used for half-life extension of small protein therapeutics. Forexample, insulin determir (LEVEMIR), an approved product for diabetes,comprises a myristyl chain conjugated to a genetically-modified insulin,resulting in a long-acting insulin analog.

The present disclosure also contemplates fusion proteins which comprisean albumin binding domain (ARD) polypeptide sequence and the sequence ofone or more of the polypeptides described herein. Any ABD polypeptidesequence described in the literature can be a component of the fusionproteins. The components of the fusion proteins can be optionallycovalently bonded through a linker, such as those linkers describedherein. In some of the embodiments of the present disclosure, the fusionproteins comprise the ABD polypeptide sequence as an N-terminal moietyand the polypeptides described herein as a C-terminal moiety.

The present disclosure also contemplates fusion proteins comprising afragment of an albumin binding polypeptide, which fragment substantiallyretains albumin binding; or a multimer of albumin binding polypeptidesor their fragments comprising at least two albumin binding polypeptidesor their fragments as monomer units. For a general discussion of ABD andrelated technologies, see WO 20121050923, WO 2012/050930, WO 2012/004384and WO 2009/016043.

Conjugation with Other Molecules:

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; tetanus toxoid; Diphtheria toxoid; polyaminoacids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides ofrotaviruses; influenza virus hemaglutinin, influenza virusnucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B viruscore protein and surface antigen; or any combination of the foregoing.

Thus, the present disclosure contemplates conjugation of one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another polypeptide (e.g., a polypeptidehaving an amino acid sequence heterologous to the subject polypeptide),or a carrier molecule. Thus, an exemplary polypeptide sequence can beprovided as a conjugate with another component or molecule.

A conjugate modification may result in a polypeptide sequence thatretains activity with an additional or complementary function oractivity derived from the second molecule. For example, a polypeptidesequence may be conjugated to a molecule, e.g., to facilitatesolubility, storage, in vivo or shelf half-life or stability, reductionin immunogenicity, delayed or controlled release in vivo, etc. Otherfunctions or activities include a conjugate that reduces toxicityrelative to an unconjugated polypeptide sequence, a conjugate thattargets a type of cell or organ more efficiently than an unconjugatedpolypeptide sequence, or a drug to further counter the causes or effectsassociated with a disease, disorder or condition as set forth herein(e.g., cancer).

An IL-10 polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, or cellulose beads; polymeric amino acids such aspolyglutamic acid, or polylysine; amino acid copolymers; inactivatedvirus particles; inactivated bacterial toxins such as toxoid fromdiphtheria, tetanus, cholera, or leukotoxin molecules; inactivatedbacteria; and dendritic cells. Such conjugated forms, if desired, can beused to produce antibodies against a polypeptide of the presentdisclosure.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH ˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from about 3 to 5.5, e.g., at pH ˜4.5. Thecontent of the fractions obtained by cation exchange chromatography maybe identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

Fc-Fusion Molecules:

In certain embodiments, the amino- or carboxyl-terminus of a polypeptidesequence of the present disclosure can be fused with an immunoglobulinFc region (e.g., human Fc) to form a fusion conjugate (or fusionmolecule). Fc fusion conjugates have been shown to increase the systemichalf-life of biopharmaceuticals, and thus the biopharmaceutical productmay require less frequent administration. Fc binds to the neonatal Fcreceptor (FcRn) in endothelial cells that line the blood vessels, and,upon binding, the Fc fusion molecule is protected from degradation andre-released into the circulation, keeping the molecule in circulationlonger. This Fc binding is believed to be the mechanism by whichendogenous IgG retains its long plasma half-life. More recent Fc-fusiontechnology links a single copy of a biopharmaceutical to the Fc regionof an antibody to optimize the pharmacokinetic and pharmacodynamicproperties of the biopharmaceutical as compared to traditional Fc-fusionconjugates. Examples of other Fc-related technologies suitable for usewith the polypeptides disclosed herein are described in WO 2013/113008.

Other Modifications:

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of IL-10 to improve one ormore properties. One such method for prolonging the circulationhalf-life, increasing the stability, reducing the clearance, or alteringthe immunogenicity or allergenicity of a polypeptide of the presentdisclosure involves modification of the polypeptide sequences byhcsylation, which utilizes hydroxyethyl starch derivatives linked toother molecules in order to modify the polypeptide sequences'characteristics. Various aspects of hesylation are described in, forexample, U.S. Patent Appln. Nos. 2007/0134197 and 2006/0258607.

The present disclosure also contemplates fusion molecules comprisingSUMO as a fusion tag (LifeSensors, Inc.; Malvern, Pa.). Fusion of apolypeptide described herein to SUMO may convey several beneficialeffects, including enhancement of expression, improvement in solubility,and/or assistance in the development of purification methods. SUMOproteases recognize the tertiary structure of SUMO and cleave the fusionprotein at the C-terminus of SUMO, thus releasing a polypeptidedescribed herein with the desired N-terminal amino acid.

Linkers:

Linkers and their use have been described above. Any of the foregoingcomponents and molecules used to modify the polypeptide sequences of thepresent disclosure may optionally be conjugated via a linker. Suitablelinkers include “flexible linkers” which are generally of sufficientlength to permit some movement between the modified polypeptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30, 30-50 or more than 50 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (for example, (GS)_(n), GSGGS_(n), GGGS_(n),(G_(m)S)_(n), (G_(m)S_(o)G_(m))_(n), (G_(m)S_(o)G_(m)S_(o)G_(m))_(n),(GSGGS_(m))_(n), (GSGS_(m)G), and (GGGS_(m))_(n), and combinationsthereof, where m, and o are each independently selected from an integerof at least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers. Glycine and glycine-serine polymers arerelatively unstructured, and therefore may serve as a neutral tetherbetween components. Exemplary flexible linkers include, but are notlimited to GGSG, GGSGG, GSGSG, GSGGG, GGGSG, and GSSSG.

Therapeutic and Prophylactic Uses

The present disclosure contemplates the use of the IL-10 polypeptidesdescribed herein (e.g., PEG-IL-10) in the treatment or prevention of abroad range of diseases, disorders and/or conditions, and/or thesymptoms thereof. Indeed, the teachings of the present disclosure aremeant to apply to any disease, disorder or condition for which achievingor maintaining the above-described IL-10 mean serum trough concentrationparameters may be beneficial. While particular uses are described indetail hereafter, it is to be understood that the present disclosure isnot so limited. Furthermore, although general categories of particulardiseases, disorders and conditions are set forth hereafter, some of thediseases, disorders and conditions may be a member of more than onecategory (e.g., cancer- and fibrotic-related disorders), and others maynot be a member of any of the disclosed categories.

Fibrotic Disorders and Cancer.

In accordance with the present disclosure, IL-10 (e.g., PEG-IL-10) canbe used to treat or prevent a proliferative condition or disorder,including a cancer (e.g., cancer of the uterus, cervix, breast,prostate, testes, gastrointestinal tract (e.g., esophagus, oropharynx,stomach, small or large intestines, colon, or rectum), kidney, renalcell, bladder, bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid,brain (e.g., gliomas), ganglia, central nervous system (CNS) andperipheral nervous system (PNS), and cancers of the hematopoietic systemand the immune system (e.g., spleen or thymus). The present disclosurealso provides methods of treating or preventing other cancer-relateddiseases, disorders or conditions, including, for example, immunogenictumors, non-immunogenic tumors, dormant tumors, virus-induced cancers(e.g., epithelial cell cancers, endothelial cell cancers, squamous cellcarcinomas and papillomavirus), adenocarcinomas, lymphomas, carcinomas,melanomas, leukemias, myelomas, sarcomas, teratocarcinomas,chemically-induced cancers, metastasis, and angiogenesis. The disclosurecontemplates reducing tolerance to a tumor cell or cancer cell antigen,e.g., by modulating activity of a regulatory T cell and/or a CD8+ T cell(see, e.g., Ramirez-Montagut, et al. (2003) Oncogene 22:3180-3187; andSawaya, et al. (2003) New Engl. J. Med. 349:1501-1509). In particularembodiments, the tumor or cancer is colon cancer, ovarian cancer, breastcancer, melanoma, lung cancer, glioblastoma, or leukemia. The use of theterm(s) cancer-related diseases, disorders and conditions is meant torefer broadly to conditions that are associated, directly or indirectly,with cancer, and includes, e.g., angiogenesis and precancerousconditions such as dysplasia.

In some embodiments, the present disclosure provides methods fortreating a proliferative condition, cancer, tumor, or precancerouscondition with an IL-10 polypeptide (e.g., PEG-IL-10) and at least oneadditional therapeutic or diagnostic agent, examples of which are setforth elsewhere herein.

The present disclosure also provides methods of treating or preventingfibrotic diseases, disorders and conditions. As used herein, the phrase“fibrotic diseases, disorders and conditions”, and similar terms (e.g.,“fibrotic disorders”) and phrases, is to be construed broadly such thatit includes any condition which may result in the formation of fibrotictissue or scar tissue (e.g., fibrosis in one or more tissues). By way ofexample, injuries (e.g., wounds) that may give rise to scar tissueinclude wounds to the skin, eye, lung, kidney, liver, central nervoussystem, and cardiovascular system. The phrase also encompasses scartissue formation resulting from stroke, and tissue adhesion, forexample, as a result of injury or surgery.

As used herein the term “fibrosis” refers to the formation of fibroustissue as a reparative or reactive process, rather than as a normalconstituent of an organ or tissue. Fibrosis is characterized byfibroblast accumulation and collagen deposition in excess of normaldeposition in any particular tissue.

Fibrotic disorders include, but are not limited to, fibrosis arisingfrom wound healing, systemic and local scleroderma, atherosclerosis,restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonaryfibrosis, interstitial lung disease, liver cirrhosis, fibrosis as aresult of chronic hepatitis B or C infection, kidney disease (e.g.,glomerulonephritis), heart disease resulting from scar tissue, keloidsand hypertrophic scars, and eye diseases such as macular degeneration,and retinal and vitreal retinopathy. Additional fibrotic diseasesinclude chemotherapeutic drug-induced fibrosis, radiation-inducedfibrosis, and injuries and burns.

Fibrotic disorders are often hepatic-related, and there is frequently anexus between such disorders and the inappropriate accumulation of livercholesterol and triglycerides within the hepatocytes. This accumulationappears to result in a pro-inflammatory response that leads to liverfibrosis and cirrhosis. Hepatic disorders having a fibrotic componentinclude non-alcoholic fatty liver disease (NAFLD) and non-alcoholicsteatohepatitis (NASH).

Cardiovascular Diseases.

The present disclosure also contemplates the use of the IL-10polypeptides (e.g., PEG-IL-10) described herein to treat and/or preventcertain cardiovascular- and/or associated metabolic-related diseases,disorders and conditions, as well as disorders associated therewith.

As used herein, the terms “cardiovascular disease”, “heart disease” andthe like refer to any disease that affects the cardiovascular system,primarily cardiac disease, vascular diseases of the brain and kidney,and peripheral arterial diseases. Cardiovascular disease is aconstellation of diseases that includes coronary heart disease (i.e.,ischemic heart disease or coronary artery disease), atherosclerosis,cardiomyopathy, hypertension, hypertensive heart disease, corpulmonale,cardiac dysrhythmias, endocarditis, cerebrovascular disease, andperipheral arterial disease. Cardiovascular disease is the leading causeof deaths worldwide, and while it usually affects older adults, theantecedents of cardiovascular disease, notably atherosclerosis, begin inearly life.

Particular embodiments of the present disclosure are directed to the useof IL-10 polypeptides to treat and/or prevent atherosclerosis, a chroniccondition in which an artery wall thickens to form plaques as a resultof the accumulation of fatty materials such as cholesterol andtriglycerides. Atherosclerosis frequently involves a chronicinflammatory response in the walls of arteries, caused largely by theaccumulation of macrophages and promoted by low-density lipoproteins(LDL) without adequate removal of fats and cholesterol from themacrophages by functional high-density lipoproteins. Chronicallyexpanding atherosclerotic lesions can cause complete closure of thelumen, which may only manifest when the lumen stenosis is so severe thatblood supply to downstream tissue(s) is insufficient, resulting inischemia.

The IL-10 polypeptides may be particularly advantageous in the treatmentand/or prevention of cholesterol-related disorders, which may beassociated with, for example, cardiovascular disease (e.g.atherosclerosis), cerebrovascular disease (e.g., stroke), and peripheralvascular disease. By way of example, but not limitation, the IL-10polypeptides may be used for lowering a subject's blood cholesterollevel. In determining whether a subject has hypercholesterolemia, thereis no firm demarcation between normal and abnormal cholesterol levels,and interpretation of values needs to be made in relation to otherhealth conditions and risk factors. Nonetheless, the followingguidelines are generally used in the United States: total cholesterol<200 mg/dL is desirable, 200-239 mg/dL is borderline high, and ≥240mg/dL is high. Higher levels of total cholesterol increase the risk ofcardiovascular disease, and levels of LDL or non-HDL cholesterol areboth predictive of future coronary heart disease. When assessinghypercholesterolemia, it is frequently useful to measure all lipoproteinsubfractions (VLDL, IDL, LDL and HDL). A particular therapeutic goal isto decrease LDL while maintaining or increasing HDL.

Thrombosis and Thrombotic Conditions.

Thrombosis, the formation of a thrombus (blood clot) inside a bloodvessel resulting in obstruction of the flow of blood through thecirculatory system, may be caused by abnormalities in one or more of thefollowing (Virchow's triad): hypercoagulability or increased bloodclotting, endothelial cell injury, or disturbed blood flow (stasis,turbulenee).

Thrombosis is generally categorized as venous or arterial, each of whichcan be presented by several subtypes. Venous thrombosis includes deepvein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis,jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease,and cerebral venous sinus thrombosis. Arterial thrombosis includesstroke and myocardial infarction.

Other diseases, disorders and conditions are contemplated by the presentdisclosure, including atrial thrombosisand Polycythemia vera (also knownas erythema, primary polycythemia and polycythemia rubra vera), amyeloproliferative blood disorder in which the bone marrow makes toomany RBCs, WBCs and/or platelets.

Immune and Inflammatory Conditions.

As used herein, terms such as “immune disease”, “immune condition”,“immune disorder”, “inflammatory disease”, “inflammatory condition”,“inflammatory disorder” and the like are meant to broadly encompass anyimmune- or inflammatory-related condition (e.g., pathologicalinflammation and autoimmune diseases). Such conditions frequently areinextricably intertwined with other diseases, disorders and conditions.By way of example, an “immune condition” may refer to proliferativeconditions, such as cancer, tumors, and angiogenesis, includinginfections (acute and chronic), tumors, and cancers that resisteradication by the immune system.

A non-limiting list of immune- and inflammatory-related diseases,disorders and conditions which may, for example, be caused byinflammatory cytokines, include, arthritis, kidney failure, lupus,asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, surgicalcomplications (e.g., where inflammatory cytokines prevent healing),anemia, and fibromyalgia. Other diseases and disorders which may beassociated with chronic inflammation include Alzheimer's disease,congestive heart failure, stroke, aortic valve stenosis,arteriosclerosis, osteoporosis, Parkinson's disease, infections,inflammatory bowel disease (e.g., Crohn's disease and ulcerativecolitis), allergic contact dermatitis and other cczemas, systemicsclerosis, transplantation and multiple sclerosis.

Some of the aforementioned diseases, disorders and conditions for whichIL-10 (e.g., PEG-IL-10) may be particularly efficacious (due to, forexample, limitations of current therapies) are described in more detailhereafter.

The IL-10 polypeptides of the present disclosure may be particularlyeffective in the treatment and prevention of inflammatory bowel diseases(IBD). IBD comprises Crohn's disease (CD) and ulcerative colitis (UC),both of which are idiopathic chronic diseases that can affect any partof the gastrointestinal tract, and are associated with many untowardeffects, and patients with prolonged UC are at an increased risk ofdeveloping colon cancer. Current IBD treatments are aimed at controllinginflammatory symptoms, and while certain agents (e.g., corticosteroids,aminosalicylates and standard immunosuppressive agents (e.g.,cyclosporine, azathioprine, and methotrexate)) have met with limitedsuccess, long-term therapy may cause liver damage (e.g., fibrosis orcirrhosis) and bone marrow suppression, and patients often becomerefractory to such treatments.

Psoriasis, a constellation of common immune-mediated chronic skindiseases, affects more than 4.5 million people in the U.S., of which 1.5million are considered to have a moderate-to severe form of the disease.Moreover, over 10% of patients with psoriasis develop psoriaticarthritis, which damages the bone and connective tissue around thejoints. An improved understanding of the underlying physiology ofpsoriasis has resulted in the introduction of agents that, for example,target the activity of T lymphocytes and cytokines responsible for theinflammatory nature of the disease. Such agents include the TNF-αinhibitors (also used in the treatment of rheumatoid arthritis (RA)),including ENBREL (etanercept), REMICADE (infliximab) and HUMIRA(adalimumab)), and T-cell inhibitors such as AMEVIVE (alefacept) andRAPTIVA (efalizumab). Though several of these agents are effective tosome extent in certain patient populations, none have been shown toeffectively treat all patients.

Rheumatoid Arthritis (RA), which is generally characterized by chronicinflammation in the membrane lining (the synovium) of the joints,affects approximately 1% of the U.S. population, or 2.1 million peoplein the U.S. Further understanding of the role of cytokines, includingTNF-α and IL-1, in the inflammatory process has enabled the developmentand introduction of a new class of disease-modifying antirheumatic drugs(DMARDs). Agents (some of which overlap with treatment modalities forRA) include ENBREL (etancrcept), REMICADE (infliximab), HUMIRA(adalimumab) and KINERET (anakinra). Though some of these agents relievesymptoms, inhibit progression of structural damage, and improve physicalfunction in particular patient populations, there is still a need foralternative agents with improved efficacy, complementary mechanisms ofaction, and fewer/less severe adverse effects.

Subjects suffering from multiple sclerosis (MS), a seriouslydebilitating autoimmune disease comprising multiple areas ofinflammation and scarring of the myelin in the brain and spinal cord,may be particularly helped by the 11-10 polypeptides described herein,as current treatments only alleviate symptoms or delay the progressionof disability.

Similarly, the IL-10 polypeptides may be particularly advantageous forsubjects afflicted with neurodegenerative disorders, such as Alzheimer'sdisease (AD), a brain disorder that seriously impairs patients' thought,memory, and language processes, and Parkinson's disease (PD), aprogressive disorder of the CNS characterized by, for example, abnonnalmovement, rigidity and tremor. These disorders are progressive anddebilitating, and no curative agents are available.

Viral Diseases.

There has been increased interest in the role of IL-10 in viraldiseases. IL-10 has been postulated to produce both stimulatory andinhibitory effects depending on its receptor binding activity.

For example, the effect of inhibiting IL-10 function in order toincrease antiviral immunity and vaccine efficacy has been considered(see Wilson, E., (201) Curr Top Microbiol Immunol. 350: 39-65).Moreover, the role of IL-10 in human immunodeficiency virus (HIV)function has been studied. In addition to the inhibition of humanimmunodeficiency virus type 1 (HIV-1) replication, IL-10 may alsopromote viral persistence by inactivation of effector immune mechanisms(Naicker, D., et al., (2009) J Infect Dis. 200 (3):448-452). Anotherstudy has identified an IL-10-producing subset of B cells able toregulate T cell immunity in chronic hepatitis B virus (HBV) infection.

Although the aforementioned studies indicate that IL-10 inhibition maybe beneficial, particular viral infections that comprise a CD8 IT cellcomponent may be candidates for treatment and/or prevention through theadministration of IL-10. This is supported by the positive role thatIL-10 plays in certain cancers by modulation of regulatory T cellsand/or CD8+ T cells.

The present disclosure contemplates the use of the IL-10 polypeptides inthe treatment and/or prevention of any viral disease, disorder orcondition for which treatment with IL-10 may be beneficial. Examples ofviral diseases, disorders and conditions that are contemplated includehepatitis B, hepatitis C, HIV, herpes virus and cytomegalovirus (CMV).

Pharmaceutical Compositions

The IL-10 polypeptides of the present disclosure may be in the form ofcompositions suitable for administration to a subject. In general, suchcompositions are “pharmaceutical compositions” comprising IL-10 and oneor more pharmaceutically acceptable or physiologically acceptablediluents, carriers or excipients. In certain embodiments, the IL-10polypeptides are present in a therapeutically acceptable amount. Thepharmaceutical compositions may be used in the methods of the presentdisclosure; thus, for example, the pharmaceutical compositions can beadministered ex vivo or in vivo to a subject in order to practice thetherapeutic and prophylactic methods and uses described herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds as describedherein in order to treat or prevent the diseases, disorders andconditions as contemplated by the present disclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of an IL-10 polypeptide contemplated by the presentdisclosure and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, detergents,buffers, vehicles, diluents, and/or adjuvants. For example, a suitablevehicle may be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers that canbe used in the pharmaceutical compositions and dosage forms contemplatedherein. Typical buffers include, but are not limited to,pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.As an example, the buffer components can be water soluble materials suchas phosphoric acid, tartaric acids, lactic acid, succinic acid, citricacid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, andsalts thereof. Acceptable buffering agents include, for example, a Trisbuffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminoprpanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus may be used to deliver IL-10,including implants (e.g., implantable pumps) and catheter systems, slowinjection pumps and devices, all of which are well known to the skilledartisan. Depot injections, which are generally administeredsubcutaneously or intramuscularly, may also be utilized to release thepolypeptides disclosed herein over a defined period of time. Depotinjections are usually either solid- or oil-based and generally compriseat least one of the formulation components set forth herein. One ofordinary skill in the art is familiar with possible formulations anduses of depot injections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or

oleagenous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents mentioned herein. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Acceptable diluents, solvents and dispersion mediathat may be employed include water, Ringer's solution, isotonic sodiumchloride solution, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphatebuffered saline (PBS), ethanol, polyol (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), and suitable mixtures thereof.In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed, including synthetic mono- or diglycerides. Moreover, fattyacids such as oleic acid, find use in the preparation of injectables.Prolonged absorption of particular injectable formulations can beachieved by including an agent that delays absorption (e.g., aluminummonostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, capsules,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups, solutions,microbeads or elixirs. Pharmaceutical compositions intended for oral usemay be prepared according to any method known to the art for themanufacture of pharmaceutical compositions, and such compositions maycontain one or more agents such as, for example, sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tablets,capsules and the like contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally occurring gums, for example, gum acacia or gumtragacanth; naturally occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed.

The present disclosure contemplates the administration of the IL-10polypeptides in the form of suppositories for rectal administration. Thesuppositories can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include, but are not limited to,cocoa butter and polyethylene glycols.

The IL-10 polypeptides contemplated by the present disclosure may be inthe form of any other suitable pharmaceutical composition (e.g., spraysfor nasal or inhalation use) currently known or developed in the future.

The concentration of a polypeptide or fragment thereof in a formulationcan vary widely (e.g., from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight) and will usually beselected primarily based on fluid volumes, viscosities, andsubject-based factors in accordance with, for example, the particularmode of administration selected.

Routes of Administration

The present disclosure contemplates the administration of IL-10, andcompositions thereof, in any appropriate manner. Suitable routes ofadministration include parenteral (e.g., intramuscular, intravenous,subcutaneous (e.g., injection or implant), intraperitoneal,intracistemal, intraarticular, intraperitoneal, intracerebral(intraparenchymal) and intracerebroventricular), oral, nasal, vaginal,sublingual, intraocular, rectal, topical (e.g., transdermal), sublingualand inhalation. Depot injections, which are generally administeredsubcutaneously or intramuscularly, may also be utilized to release theIL-10 polypeptides disclosed herein over a defined period of time.

Particular embodiments of the present disclosure contemplate parenteraladministration, and in further particular embodiments the parenteraladministration is subcutaneous.

Combination Therapy

The present disclosure contemplates the use of IL-10 (e.g., PEG-IL-10)in combination with one or more active therapeutic agents (e.g.,cytokines) or other prophylactic or therapeutic modalities (e.g.,radiation). In such combination therapy, the various active agentsfrequently have different mechanisms of action. Such combination therapymay be especially advantageous by allowing a dose reduction of one ormore of the agents, thereby reducing or eliminating the adverse effectsassociated with one or more of the agents; furthermore, such combinationtherapy may have a synergistic therapeutic or prophylactic effect on theunderlying disease, disorder, or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, the IL-10 polypeptides are administered orapplied sequentially, e.g., where one agent is administered prior to oneor more other agents. In other embodiments, the IL-10 polypeptides areadministered simultaneously, e.g., where two or more agents areadministered at or about the same time; the two or more agents may bepresent in two or more separate formulations or combined into a singleformulation (i.e., a co-formulation). Regardless of whether the two ormore agents are administered sequentially or simultaneously, they areconsidered to be administered in combination for purposes of the presentdisclosure.

The IL-10 polypeptides of the present disclosure may be used incombination with one or more other (active) agent in any mannerappropriate under the circumstances. In one embodiment, treatment withthe at least one active agent and at least one IL-10 polypeptide of thepresent disclosure is maintained over a period of time. In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe IL-10 polypeptide of the present disclosure is maintained at aconstant dosing regimen. In a further embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the IL-10 polypeptide of thepresent disclosure is reduced (e.g., lower dose, less frequent dosing orshorter treatment regimen). In yet another embodiment, treatment withthe at least one active agent is reduced or discontinued (e.g., when thesubject is stable), and treatment with the IL-10 polypeptide of thepresent disclosure is increased (e.g., higher dose, more frequent dosingor longer treatment regimen). In yet another embodiment, treatment withthe at least one active agent is maintained and treatment with the IL-10polypeptide of the present disclosure is reduced or discontinued (e.g.,lower dose, less frequent dosing or shorter treatment regimen). In yetanother embodiment, treatment with the at least one active agent andtreatment with the IL-10 polypeptide of the present disclosure arereduced or discontinued (e.g., lower dose, less frequent dosing orshorter treatment regimen).

Fibrotic Disorders and Cancer.

The present disclosure provides methods for treating and/or preventing aproliferative condition, cancer, tumor, or precancerous disease,disorder or condition with an IL-10 polypeptide (e.g., PEG-IL-10) and atleast one additional therapeutic or diagnostic agent.

Examples of chemotherapeutic agents include, but are not limited to,alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretarnine, triethyleneielamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamime; nitrogen mustards such as chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofobsfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum and platinum coordinationcomplexes such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitors;difluoromethylomithinc (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Chemotherapeutic agents also include anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens,including for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imrnidazoles, 4-hydroxytamoxifen, trioxifene, keuxifene,onapristone, and toremifene; and antiandrogens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In certain embodiments, combination therapy comprisesadministration of a hormone or related hormonal agent.

Additional treatment modalities that may be used in combination with theIL-10 polypeptides include a cytokine or cytokine antagonist, such asIL-12, INFα, or anti-epidermal growth factor receptor, radiotherapy, amonoclonal antibody against another tumor antigen, a complex of amonoclonal antibody and toxin, a T-cell adjuvant, bone marrowtransplant, or antigen presenting cells (e.g., dendritic cell therapy).Vaccines (e.g., as a soluble protein or as a nucleic acid encoding theprotein) are also provided herein.

Cardiovascular Diseases.

The present disclosure provides methods for treating and/or preventingcertain cardiovascular- and/or metabolic-related diseases, disorders andconditions, as well as disorders associated therewith, with an IL-10polypeptide (e.g., PEG-IL-10) and at least one additional therapeutic ordiagnostic agent.

Examples of therapeutic agents useful in combination therapy for thetreatment of hypercholesterolemia (and thus frequently atherosclerosis)include statins (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR, PRAVACOL, andZOCOR), which inhibit the enzymatic synthesis of cholesterol; bile acidresins (e.g., COLESTID, LO-CHOLEST, PREVALITE, QUESTRAN, and WELCHOL),which sequester cholesterol and prevent its absorption; ezetimibe(ZETIA), which blocks cholesterol absorption; fibric acid (e.g.,TRICOR), which reduce triglycerides and may modestly increase HDL;niacin (e.g., NIACOR), which modestly lowers LDL cholesterol andtriglycerides; and/or a combination of the aforementioned (e.g., VYTORIN(czctimibe with simvastatin). Alternative cholesterol treatments thatmay be candidates for use in combination with the IL-10 polypeptidesdescribed herein include various supplements and herbs (e.g., garlic,policosanol, and guggul). The present disclosure encompassespharmaceutically acceptable salts, acids or derivatives of any of theabove.

Immune and Inflammatory Conditions.

The present disclosure provides methods for treating and/or preventingimmune- and/or inflammatory-related diseases, disorders and conditions,as well as disorders associated therewith, with an IL-10 polypeptide(e.g., PEG-IL-10) and at least one additional therapeutic or diagnosticagent.

Examples of therapeutic agents useful in combination therapy include,but are not limited to, the following: non-steroidal anti-inflammatorydrug (NSAID) such as aspirin, ibuprofen, and other propionic acidderivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen,fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen,miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen,tiaprofenic acid, and tioxaprofen), acetic acid derivatives(indomethacin, acemetacin, alclofenac, clidanac, diclofenac,fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac,oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac),fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamicacid, niflumic acid and tolfenamic acid), biphenylcarboxylic acidderivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam,sudoxicarn and tenoxican), salicylates (acetyl salicylic acid,sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone,mofebutazone, oxyphenbutazone, phenylbutazone). Other combinationsinclude cyclooxygenase-2 (COX-2) inhibitors.

Other active agents for combination include steroids such asprednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, or hydrocortisone. Such a combination may be especiallyadvantageous, since one or more side effects of the steroid can bereduced or even eliminated by tapering the steroid dose required whentreating patients in combination with the present IL-10 polypeptides.

Additional examples of active agents for combinations for treating, forexample, rheumatoid arthritis include cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists ofother human cytokines or growth factors, for example, TNF, LT, IL-1β,IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, orPDGF.

Particular combinations of active agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade, andinclude TNF antagonists like chimeric, humanized or human TNFantibodies. REMICADE, anti-TNF antibody fragments (e.g., CDP870), andsoluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG(ENBREL) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor (sIL-13), andalso TNFα converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors(e.g., Interleukin-1-converting enzyme inhibitors) may be effective.Other combinations include Interleukin 11, anti-P7s and p-selectinglycoprotein ligand (PSGL). Other examples of agents useful incombination with the IL-10 polypeptides described herein includeinterferon-β1a (AVONEX); interferon-β1b (BETASERON); copaxone;hyperbaric oxygen; intravenous immunoglobulin; clabribine; andantibodies to or antagonists of other human cytokines or growth factors(e.g., antibodies to CD40 ligand and CD80).

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Viral Diseases.

The present disclosure provides methods for treating and/or preventingviral diseases, disorders and conditions, as well as disordersassociated therewith, with an IL-10 polypeptide (e.g., PEG-IL-10) and atleast one additional therapeutic or diagnostic agent (e.g., one or moreother anti-viral agents and/or one or more other non-viral agents).

Such combination therapy includes anti-viral agents targeting variousviral life-cycle stages and having different mechanisms of action,including, but not limiting to, the following: inhibitors of viraluncoating (e.g., amantadine and rimantidine); reverse transcriptaseinhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents thattarget integrase; agents that block attachment of transcription factorsto viral DNA; agents (e.g., antisense molecules) that impact translation(e.g., fomivirsen); agents that modulate translation/ribozyme function;protease inhibitors; viral assembly modulators (e.g., rifampicin); andagents that prevent release of viral particles (e.g., zanamivir andoseltamivir). Treatment and/or prevention of certain viral infections(e.g., HIV) frequently entail a group (“cocktail”) of antiviral agents.

Other antiviral agents contemplated for use in combination with IL-10polypeptides include, but are not limited to, the following: abacavir,adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir,atripla, boceprevirertet, cidofbvir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, ganciclovir,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,various interferons (e.g., peginterferon alfa-2a), lopinavir, loviride,maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir,penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir,ribavirin, ritonavir, pyramidinc, saquinavir, stavudinc, telaprcvir,tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada,valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, andzalcitabine.

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Dosing

The IL-10 polypeptides of the present disclosure may be administered toa subject in an amount that is dependent upon, for example, the goal ofthe administration (e.g., the degree of resolution desired); the age,weight, sex, and health and physical condition of the subject theformulation being administered; the route of administration; and thenature of the disease, disorder, condition or symptom thereof. Thedosing regimen may also take into consideration the existence, nature,and extent of any adverse effects associated with the agent(s) beingadministered. Effective dosage amounts and dosage regimens can readilybe determined from, for example, safety and dose-escalation trials, invivo studies (e.g., animal models), and other methods known to theskilled artisan.

The present disclosure contemplates administration of IL-10 to achievecertain serum trough concentrations and/or maintain certain mean serumtrough concentrations. Methodologies specific to IL-10 are describedelsewhere herein and in this section below.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with ADME, taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the IL-10 polypeptide of the presentdisclosure may be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, for a subject experiencing a particular disorder, aneffective dose may be one that improves a diagnostic parameter, measure,marker and the like of that disorder by at least about 5%, at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or more than 90%,where 100% is defined as the diagnostic parameter, measure, marker andthe like exhibited by a normal subject.

The amount of PEG-IL-10 necessary to treat a disease, disorder orcondition described herein is based on the IL-10 activity of theconjugated protein, which can be determined by IL-10 activity assaysknown in the art. By way of example, in the tumor context, suitableIL-10 activity includes, for example, CD8+ T-cell infiltrate into tumorsites, expression of inflammatory cytokines, such as IFN-γ, IL-4, IL-6,IL-10, and RANK-L, from these infiltrating cells, and increased levelsof IFN-γ in biological samples.

The therapeutically effective amount of an IL-10 agent can range fromabout 0.01 to about 100 μg protein kg of body weight/day, from about 0.1to 20 μg protein/kg of body weight/day, from about 0.5 to 10 μgprotein/kg of body weight/day, or about 1 to 4 μg protein/kg of bodyweight/day. In some embodiments, an IL-10 agent is administered bycontinuous infusion to deliver about 50 to 800 μg protein/kg of bodyweight/day (e.g., about 1 to 16 μg protein/kg of body weight/day of theIL-10 agent). The infusion rate may be varied based on evaluation of,for example, adverse effects and blood cell counts.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 10 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient.

Particular dosing regimens (e.g., dosing frequencies) for the IL-10polypeptides are described elsewhere herein.

In certain embodiments, the dosage of the disclosed IL-10 polypeptide iscontained in a “unit dosage form”. The phrase “unit dosage form” refersto physically discrete units, each unit containing a predeterminedamount of a IL-10 polypeptide of the present disclosure, either alone orin combination with one or more additional agents, sufficient to producethe desired effect. It will be appreciated that the parameters of a unitdosage form will depend on the particular agent and the effect to beachieved.

Kits

The present disclosure also contemplates kits comprising IL-10, andpharmaceutical compositions thereof. The kits are generally in the formof a physical structure housing various components, as described below,and may be utilized, for example, in practicing the methods describedabove (e.g., administration of a IL-10 polypeptide to a subject in needof restoring cholesterol homeostasis).

A kit can include one or more of the IL-10 polypeptides disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. TheIL-10 polypeptides can be provided in a form that is ready for use or ina form requiring, for example, reconstitution or dilution prior toadministration. When the IL-10 polypeptides are in a form that needs tobe reconstituted by a user, the kit may also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the IL-10 polypeptides. When combination therapy iscontemplated, the kit may contain the several agents separately or theymay already be combined in the kit. Each component of the kit may beenclosed within an individual container, and all of the variouscontainers may be within a single package. A kit of the presentdisclosure may be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, tube or vial).

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below were performed and areall of the experiments that may be performed. It is to be understoodthat exemplary descriptions written in the present tense were notnecessarily performed, but rather that the descriptions can be performedto generate the data and the like described therein. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.), but some experimental errors and deviations shouldbe accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); ng=nanogram;μg=microgram; mg=milligram; g=gram; kg=kilogram; dl or dL=deciliter; Itlor tIL=microliter; ml or mL=milliliter; l or L=liter, μM=micromolar,mM=millimolar; M=molar, kDa=kilodalton; i.m.=intramuscular(ly);i.p.=intrapcritoneal(ly); i.v, or IV=intravenous(ly); s.c.orSC=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly,HPLC=high perfobrmance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PBS=phosphatc-buffcrcd saline;PCR=polymerase chain reaction; NHS=N-Hydroxysuccinimide; DMEM=Dulbeco'sModification ofEagle's Medium; GC=genome copy;EDTA=ethylenediaminetetraacetic acid.

Materials and Methods

The following general materials and methods may be used in the Examplesbelow:

Standard methods in molecular biology are described (see, e.g., Sambrookand Russell (2001) Molecular Cloning, 3′ ed., Cold Spring HarborLaboratory Press. Cold Spring Harbor. N.Y.; and Ausubel, et al. (2001)Current Protocols in Molecular Biology. Vols. 1-4, John Wiley and Sons,Inc. New York, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4)).

The scientific literature describes methods for protein purification,including immunoprecipitation, chromatography, electrophoresis,centrifugation, and crystallization, as well as chemical analysis,chemical modification, post-translational modification, production offusion proteins, and glycosylation of proteins (see, e.g., Coligan, etal. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wileyand Sons, Inc., NY).

Production, purification, and fragmentation of polyclonal and monoclonalantibodies are described (e.g., Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); standardtechniques for characterizing ligand/receptor interactions are available(see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol.4. John Wiley, Inc., NY); methods for flow cytometry, includingfluorescence-activated cell sorting (FACS), are available (see, e.g.,Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken,N.J.); and fluorescent reagents suitable for modifying nucleic acids,including nucleic acid primers and probes, polypeptides, and antibodies,for use, for example, as diagnostic reagents, are available (MolecularProbes (2003) Catalogue, Molecular Probes. Inc., Eugene, Oreg.;Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill,New York, N.Y.).

Depletion of immune cells (CD4⁺ and CD8⁺ T-cells) may be effected byantibody-mediated elimination. For example, 250 μg of CD4- orCD8-specific antibodies may be injected weekly, and cell depletionsverified using FACS and IHC analysis.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); and DeCypher™(TimeLogic Corp., Crystal Bay, Nev.).

Immunocompetent Balb/C or B-cell-deficient Balb/C mice were obtainedfrom The Jackson Lab., Bar Harbor, Me., and used in accordance withstandard procedures (see, e.g., Martin et al (2001) Infect Immun.,69(11):7067-73 and Compton et al. (2004) Comp. Med. 54(6):681-89). Othermice strains suitable for the experimental work contemplated by thepresent disclosure are known to the skilled artisan and are generallyavailable from The Jackson Lab.

Unless otherwise indicated, PDV6 squamous cell carcinoma of the skin wasused in the experiments described herein (see, e.g., Langowski et al.(2006) Nature 442:461-465). Other oncology-related models and celllines, such as Ep2 mammary carcinoma, CT26 colon carcinoma, and 4T1breast carcinoma models, may be used (see, e.g., Langowski et al. (2006)Nature 442:461-465) and are known to the skilled artisan.Non-oncology-related models and cell lines (e.g., models ofinflammation) may also be used and are known to the skilled artisan.

Serum IL-10 concentration levels and exposure levels may be determinedby standard methods used in the art. For example, a serum exposure levelassay can be performed by collecting whole blood (˜50 μl/mouse) frommouse tail snips into plain capillary tubes, separating serum and bloodcells by centrifugation, and determining IL-10 exposure levels bystandard ELISA kits and techniques. Additional means of determiningIL-10 serum concentrations are described hereafter.

Production of Pegylated IL-10

The present disclosure contemplates the synthesis of pegylated IL-10 byany means known to the skilled artisan. The description hereafter ofseveral alternative synthetic schemes for producing mono-PEG-IL-10 and amix of mono-/di-PEG-IL-10 is meant to be illustrative only. While bothmono-PEG-IL-10 and a mix of mono-/di-PEG-IL-10 have many comparableproperties, a mix of selectively pegylated mono- and di-PEG-IL-10improves the yield of the final pegylated product (see, e.g., U.S. Pat.No. 7,052,686 and US Pat. Publn. No. 2011/0250163).

In addition to leveraging her own skills in the production and use ofPEGs (and other drug delivery technologies) suitable in the practice ofthe present disclosure, the skilled artisan is also familiar with manycommercial suppliers of PEG-related technologies (and other drugdelivery technologies). By way of example, NOF America Corp (Irvine,Calif.) supplies mono-functional Linear PEGs, bi-functional PEGs,multi-arm PESs, branched PEGs, heterofunctional PEGs, forked PEGs, andreleasable PEGs; and Parchem (New Rochelle, N.Y.) is a globaldistributor of PEG products and other specialty raw materials.

Exemplary PEG-IL-10 Synthetic Scheme No. 1.

IL-10 may be dialyzed against 10 mM sodium phosphate at pH 7.0, 100 mMNaCl. The dialyzed IL-10 may then be diluted 3.2 times to aconcentration of 4 mg/mL using the dialysis buffer. Prior to theaddition of the linker, SC-PEG-12K (Delmar Scientific Labs; Maywood,Ill.), 1 volume of 100 mM Na-tetraborate at pH 9.1 can be added into 9volumes of the diluted IL-10 to raise the pH of the IL-10 solution to8.6. The SC-PEG-12K linker can be dissolved in the dialysis buffer andthe appropriate volume of the linker solution (1.8 to 3.6 mole oflinker/mole of IL-10) can be added into the diluted IL-10 solution tostart the pegylation reaction. The reaction can be carried out at 5° C.in order to control the rate of the reaction. The reaction solution canbe mildly agitated during the pegylation reaction. When themono-PEG-IL-10 yield, as determined by size exclusion HPLC (SE-HPLC), isclose to 40%, the reaction is stopped by adding 1M glycine solution to afinal concentration of 30 mM. The pH of the reaction solution is slowlyadjusted to 7.0 using an HCl solution, and the reaction solution is thenfiltered using a 0.2 micron filter and stored at −80° C.

Exemplary PEG-IL-10 Synthetic Scheme No. 2.

Mono-PEG-IL-10 is prepared using methoxy-PEG-aldehyde (PALD-PEG) as alinker (Inhale Therapeutic Systems Inc., Huntsville, Ala.; alsoavailable from NOF America Corp (Irvine, Calif.)). PALD-PEG can havemolecular weights of 5 KDa. 12 KDa, or 20 KDa. IL-10 is dialyzed anddiluted as described above, except the pH of the reaction buffer isbetween 6.3 and 7.5. Activated PALD-PEG linker is added to reactionbuffer at a 1:1 molar ratio. Aqueous cyanoborohydride is added to thereaction mixture to a final concentration of 0.5 to 0.75 mM. Thereaction is carried out at room temperature (18-25° C.) for 15-20 hourswith mild agitation. The reaction is quenched with 1M glycine. Yieldsare analyzed by SE-HPLC. Mono-PEG-IL-10 is separated from unreactedIL-10, PEG linker and di-PEG-IL-10 by gel filtration chromatography andcharacterized by RP-HPLC and bioassay (e.g., stimulation ofIL-10-responsive cells or cell lines).

Exemplary PEG-IL-10 Synthetic Scheme No. 3

IL-10 (e.g., rodent or primate) is dialyzed against 50 mM sodiumphosphate, 100 mM sodium chloride pH ranges 5-7.4. A 1:1-1:7 molar ratioof 5K PEG-propylaldehyde is reacted with IL-10 at a concentration of1-12 mg/mL in the presence of 0.75-30 mM sodium cyanoborohydride.Alternatively the reaction can be activated with picoline borane in asimilar manner. The reaction is incubated at 5-30° C. for 3-24 hours.

The pH of the pegylation reaction is adjusted to 6.3, 7.5 mg/mL ofhIL-10 is reacted with PEG to make the ratio of IL-10 to PEG linker1:3.5. The final concentration of cyanoborohydride is ˜25 mM, and thereaction is carried out at 15° C. for 12-15 hours. The mono- and di-PEGIL-10 are the largest products of the reaction, with the concentrationof each at ˜45-50% at termination. The reaction may be quenched using anamino acid such as glycine or lysine or, alternatively. Tris buffers.Multiple purification methods can be employed such as gel filtration,anion and cation exchange chromatographies, and size exclusion HPLC(SE-HPLC) to isolate the desired pegylated IL-10 molecules.

Exemplary PEG-IL-10 Synthetic Scheme No. 4.

IL-10 is dialyzed against 10 mM sodium phosphate pH 7.0, 100 mM NaCl.The dialyzed IL-10 is diluted 3.2 times to a concentration of about 0.5to 12 mg/mL using the dialysis buffer. Prior to the addition of thelinker, SC-PEG-12K (Delmar Scientific Laboratories, Maywood, Ill.), onevolume of 100 mM Na-tetraborate at pH 9.1 is added into 9 volumes of thediluted IL-10 to raise the pH of the IL-10 solution to 8.6. TheSC-PEG-12K linker is dissolved in the dialysis buffer and theappropriate volume of the linker solution (1.8 to 3.6 mole linker permole of IL-10) is added into the diluted IL-10 solution to initiate thepegylation reaction. The reaction is carried out at 5° C. in order tocontrol the rate of the reaction, and the reaction solution is mildlyagitated. When the mono-PEG-IL-10 yield, as determined by size exclusionHPLC (SE-HPLC), is close to 40%, the reaction is stopped by adding 1Mglycine solution to a final concentration of 30 mM. The pH of thereaction solution is slowly adjusted to 7.0 using an HCl solution, andthe reaction is 0.2 micron filtered and stored at −80° C.

The material set forth below, up to and including Table 15 and thedescription thereof, was essentially extracted from US Pat. Publn. No.2011/00911419 (a co-inventor of US Pat. Publn. No. 2011/00911419 is alsoan inventor of the instant application), and the teachings therein, andvariations thereof, are broadly applicable and can be utilized and/ormodified in a number of different contexts. Similarly, the teachings ofother publications in related fields and/or technology areas (see, e.g.,U.S. Pat. Nos. 6,387,364 and 7,052,684, and PCT Publn No. WO2006/075138), along with the general knowledge of the skilled artisan,can form the basis for additional experimental work.

Tumor Models and Tumor Analysis

Any art-accepted tumor model, assay, and the like can be used toevaluate the effect of IL-10 and PEG-IL-10 on various tumors. The tumormodels and tumor analyses described hereafter are representative ofthose that can be utilized, and they were used to generate and assessthe data set forth in Tables 1-15.

Syngeneic mouse tumor cells are injected subcutaneously or intradermallyat 10⁴, 10⁵ or 10⁶ cells per tumor inoculation Ep2 mammary carcinoma,CT26 colon carcinoma, PDV6 squamous carcinoma of the skin and 4T1 breastcarcinoma models can be used (see, e.g., Langowski et al. (2006) Nature442:461-465). Immunocompetent Balb/C or B cell deficient Balb/C mice canbe used. PEG-mIL-10 can be administered to the immunocompetent mice,while PEG-hIL-10 treatment can be used in the B-cell deficient mice.Tumors are allowed to reach a size of 100-250 mm before treatment isstarted. IL-10, PEG-mIL-10, PEG-hIL-10, or buffer control isadministered subcutaneously at a site distant from the tumorimplantation. Tumor growth is typically monitored twice weekly usingelectronic calipers.

Tumor tissues and lymphatic organs are harvested at various endpoints tomeasure mRNA expression for a number of inflammatory markers and toperform immunohistochemistry for several inflammatory cell markers. Thetissues are snap-frozen in liquid nitrogen and stored at −80° C. Primarytumor growth is typically monitored twice weekly using electroniccalipers. Tumor volume may be calculated using the formula(width²×length/2) where length is the longer dimension. Tumors areallowed to reach a size of 90-250 mm³ before treatment is started.

Administration of IL-10 and/or PEG-IL-10

The tumor models and tumor analysis methods described above wereutilized to generate the data set forth hereafter. However, as alludedto above, these same models and methodologies may be used in otherexperimental settings.

Murine IL-10 (mIL-10) or PEG-mIL-10 were administered to theimmunocompetent mice, while PEG-hIL-10 treatment was used in the B-celldeficient mice. Murine IL-10, PEG-mIL-10, PEG-hIL-0, or vehicle controlwas administered subcutaneously at a site distant from the tumorimplant. PEG-mIL-10 used in these studies was prepared with theSC-PEG-12K linker. The biological activities of mIL-10 and PEG-m IL-10were assessed by the application of a short-term proliferation bioassaythat utilizes MC/9, a mouse mast cell line, which expresses endogenousmIL-10 receptors (R1 and R2). The MC/9 cells proliferate in response toco-stimulation with mIL-4 and mIL-10 (MC/9 cells do not proliferate withonly mIL-4 or mIL-10). Proliferation was measured by colorimetric meansusing Alamar Blue, a growth indicator dye based on detection ofmetabolic activity. The biological activity of recombinant or PEG-mIL-10was assessed by the EC50 value, or the concentration of protein at whichhalf-maximal stimulation is observed in a dose-response curve (Table 1).

TABLE 1 MC/9 Proliferation bioassay for the assessment of bioactivity ofmIL-10 and PEG-mIL10 reagents used in these studies Protein EC50 (ng/mL)in MC/9 Assay mIL-10 0.5711 PEG-mIL-10 4.039

As indicated in Table 1, based on the MC/9 bioassay the specificactivity of the PEG-mIL-10 used in the experiments is approximately7-fold lower than the activity of the mIL-10.

PEG-mIL-10 may also be administered every second day to mice harboringEp2 breast cancer tumors. Treatment was effective in reducing tumor sizeand inducing tumor rejections.

TABLE 2 PEG mIL-10 reduces tumor size (mm³) in Ep2 breast cancer modelin Balb/C mice. Days after Inoculation 11 15 18 21 25 27 33 Control 300450 500 750 1300 1500 2700 PEG-IL-10 300 400 310 280 250 50 0

Treatment with PEG-mIL-10 was also effective in reducing tumor size inPDV6. CT-26, and 4T1 syngeneic immune competent mouse tumor models (seeTables 3, 4, and 5).

TABLE 3 Study 04-M52 338: PEG mIL-10 beginning day 36 after implantreduces PDV6 tumor size (mm²) in C57B/6 mice.. Days after Inoculation 3638 42 44 46 48 52 Control 200 255 290 380 395 420 485 PEG-mIL-10 210 265200 190 155 110 55

TABLE 4 PEG mIL-10 beginning day 7 after implant reduces tumor sizerelative to vehicle control of CT26 tumors (mm³) in BALB/c mice. Daysafter Inoculation 10 15 17 20 22 24 Vehicle Control 155 424 791 12741737 2170 PEG-mIL-10 136 212 291 336 450 455

TABLE 5 IL-10 and PEG mIL-10 reduces tumor size (mm³) of 4T1 breastcarcinoma Days of Treatment 20 24 29 33 Control 200 410 584 1000PEG-mIL-10 200 320 560 350 IL-10 200 290 575 400Dose Titration Studies

In dose titration studies, tail-vein bleeds were collected fromrepresentative mice of each group at times corresponding to the expectedpeak and trough dose levels. Serum harvested was assayed for mIL-10concentrations using the Meso Scale Discovery platform which is based onmulti-array technology, a combination of electrochemiluminescencedetection and patterned arrays. A two-tailed unpaired student t-test wasused to compare the mean tumor volume of mIL-10 or PEG-mIL-10-treatedmice grouped by serum mIL-10 concentration with the mean tumor volume oftheir corresponding vehicle control group. A Welch's correction was usedwhen two groups had unequal variance (p<0.05 from t-test).

Dose titrations of PEG-mIL-10 and mIL-10 in 4T1 breast carcinoma-bearingmice show that control of primary tumor and lung metastases aredosetitratable with both mIL-10 and with PEG-mIL-10. As set forth inTable 6, at any given dose PEG-mIL-10 is more effective than mIL-10.Twice daily treatment was started on Day 17 after implant, when the meantumor volumes were 84-90 mm³. Treatment groups consisted of 14 mice pergroup while the control groups had 8 mice in each group. Tris and Hepesbuffers were the controls for mIL-10 and PEG mL-10, respectively.

TABLE 6 Study 06-M175-1103. mIL-10 and PEG-mIL-10 reduce primary tumorsize (mm³) of 4T1breast carcinoma in BALB/c mice in a dose-dependentmanner. Days after Implant 17 21 24 27 30 34 38 42 Tris Vehicle 90 184288 448 560 861 1126 1248 control Hepes Vehicle 90 215 344 476 658 9401261 1520 control PEG-mIL-10 86 107 117 129 150 165 204 195 (0.5 mg/kg)PEG-mIL-10 84 112 142 152 224 256 286 356 (0.1 mg/kg) PEG-mIL-10 85 140200 240 288 462 627 773 (0.01 mg/kg) PEG-mIL-10 88 168 239 262 373 532729 942 (0.001 mg/kg) mIL-10 85 117 168 207 256 350 446 497 (1.0 mg/kg)mIL-10 84 136 180 251 337 424 641 704 (0.1 mg/kg) mIL-10 86 121 165 231331 436 631 809 (0.01 mg/kg)

Dose titrations of PEG-mIL-10 and mIL-10 in PDV6 squamous cellcarcinoma-bearing mice show that control of primary tumor isdosetitratable with both mIL-10 and with PEG-mIL-10, though at any givendose PEG-mIL-10 is more effective than mIL-10 (Table 7). The high dosePEG-mIL-10 treatment resulted in a near 100% tumor regression andsubsequent resistance to re-challenge (Table 8). Twice daily treatmentwas started on Day 23 after implant, when the mean tumor volumes were107-109 mm³ and continued through day 55 for all mIL10-treated groupsand 0.01 mg/kg PEG mIL-10 treated group. The 0.1 mg/kg PEG-mIL-10treatment was stopped on day 48 when 100% tumor regression was seen,while the remaining groups were treated until day 51. Treatment groupsconsisted of 10 mice per group while each vehicle control contained 6mice. Tris buffer and Hepes buffer were the vehicle control for mIL-10and PEG mIL-10, respectively. Re-implant was done 85 days after theprimary implant and 4 weeks after the last PEG-mIL10 treatment Therewere 10 mice per group.

TABLE 7 Study 06-M52-1106. mIL-10 and PEG-mIL-10 reduce tumor size (mm³)of PDV6 squamous cell carcinoma in C57B16/J mice in a dose-dependentmanner. Days after Implant 23 27 30 33 36 40 43 47 51 55 Tris Vehicle111 179 232 318 412 493 635 848 958 control Hepes Vehicle 107 210 293433 541 653 712 761 986 control PEG-mIL-10 108 99 55 31 17 11 3 1 1 1(0.1 mg/kg) PEG-mIL-10 107 131 92 97 95 114 119 123 183 228 (0.01 mg/kg)PEG-mIL-10 109 191 191 241 327 455 535 (0.001 mg/kg) mIL-10 107 129 144143 124 87 51 36 52 75 (1.0 mg/kg) mIL-10 107 85 85 88 117 121 130 143182 217 (0.1 mg/kg) mIL-10 107 120 150 146 196 244 262 263 249 250 (0.01mg/kg)

TABLE 8 Study 06-M52-1106. C57B1/6J mice that have cleared PDV6 squamouscell carcinoma tumors after 3 weeks of PEG-mIL-10 treatment areresistant to re-implant in the absence of additional treatment.. Daysafter Implant % mice that are 0 16 21 28 36 49 tumor positive Vehicle 0113 145 188 418 761 100 Control PEG-mIL-10 0 0.3 0 7 16 47 10 (0.1mg/kg)

Lung Metastasis Studies

Lung metastases in the 4T1 breast carcinoma model were either quantifiedmacroscopically after lung resection (Table 9) or by counting the lungmetastatic colonies after culture (Table 10) as described in CurrentProtocols in Immunology (Section 20.2.4) John Wiley and Sons, Inc., NewYork; Harlow and Lane (1999). Briefly, lungs harvested from a 4T1tumor-bearing mouse were minced and digested with a collagenase/elastasecocktail followed by culture in a limiting dilution assay, in mediumcontaining 6-thioguanine. Only 4T1 cells are 6-thioguanine-resistant andcan be quantified by counting the number of colonies after 10-14 days ofculture. Twice daily treatment was started on Day 17 after implant, whenthe mean tumor volumes were 84-90 mm^(J). Tris and Hepes buffers werethe controls for mIL-10 and PEG mL-10, respectively. Lung metastaseswere measured as the number of metastatic colonies cultured per lung.

TABLE 9 Study 05-M52-496. 2 week treatment with mIL-10 and PEG-mIL-10beginning 19 days after implant reduces metastasis of 4T1 breastcarcinoma (measured as number of lung metastases per mouse) LungMetastasis 33 days after Inoculation Vehicle Control mIL-10 PEG-mIL-10Mouse #1 7 0 0 Mouse #2 7 0 0 Mouse #3 7 0 0 Mouse #4 8 0 0 Mouse #5 204 0

TABLE 10 Study 06-M175-1103. mIL-10 and PEG-mIL-10 reduce lungmetastases of 4T1 breast carcinoma in BALB/c mice in a dose-dependentmanner. Lung Metastases 42-45 days after Implant Colonies per lung(×10³) Tris Hepes buffer buffer PEG- PEG- PEG- PEG- Vehicle VehiclemIL-10 mIL-10 mIL-10 mIL-10 mIL-10 mIL-10 mIL-10 Mouse control control1.0 mg/kg 0.1 mg/kg 0.01 mg/kg 0.5 mg/kg 0.1 mg/kg 0.01 mg/kg 0.001mg/kg 1 362 481 76 116 1064 7.1 89 0.43 366 2 2.12 533 20 5.6 150 1.00.7 234 212 3 152 264 28.1 8.1 67.4 0.4 0.01 377 0.6 4 0.4 218 1.2 13718 1.5 223 315 586 5 1000 517 45.7 257 77 0.3 0.07 0.54 486 6 474 9321.7 2.72 1.2 0.02 10.1 1.67 844 7 524 1000 4.4 364 285 0 7.6 68 6.5 81000 1026 128.6 772 9.7 0.002 1.85 27 265 9 13.3 348 878 0.3 0.01 139338 10 51.2 204 45 0.03 0.01 177 824 11 9.4 49 56 0.01 2.68 597 263 120.1 635 17.1 240 0.01 7.4 13 5.1 19.7 1014 0.02 2.94 0.01 14 0.02 75072.2 0.01 0.01 0.01 Median 418.0 499.0 16.7 170.5 69.8 0.17 1.28 47.5338.0 Mean 502.0 579.0 28.9 262.0 268.2 17.9 24.1 138.9 381.0 S.D. 519.0467.0 36.5 276.9 397.1 64.0 61.8 183.7 284.0

Administering PEG-mIL-10 or IL-10 to 4T1 breast carcinoma-bearing micereduces the rate of metastasis and increases CD8+ T-cell infiltrationand expression of immune stimulatory cytokines, as measured byquantitative RT-PCR (Tables 11 and 12). The number of infiltrating CD8+T-cells was counted from representative sections of several tumorsstained by immunohistochemistry for the CD8 surface marker and verifiedby staining with anti-CD3 and anti-TCRαβ antibodies.

TABLE 11 IL-10 and PEG mIL-10 induce CD8+ T-cell infiltration in 4T1carcinoma control IL-10 PEG-IL-10 Average Number of CD8+ Cells/Field 6.425.8 39.2

PEG-mIL-10 is more effective than IL-10 in the induction of inflammatorycytokines. Total RNA from homogenized tumor samples was extracted andreverse-transcribed as previously described (see, e.g., Homey, et al.(2000) J Immunol. 164:3465-3470). Complementary DNA was quantitativelyanalyzed for expression of cytokines by the fluorogenic 5′-nuclease PCRassay (see, e.g., Holland, et al. (1991) Proc. Natl. Acad. Sci.88:7276-7280). Specific PCR products were continuously measured by meansof an ABI PRISM 7700 Sequence Detection System (Applied Biosystems)during 40 cycles. Values were normalized to ubiquitin. Log-transformeddata were subjected to Kruskal-Wallis statistical analysis (medianmethod). The expression level (log transformed) corresponds to theamount of inflammatory cytokine expressed in the tumor sample, such thatthe higher the expression level (log transformed), the greater theamount of inflammatory cytokine expressed in the tumor sample.

TABLE 12 Administered PEG-mIL-10 induces sustained levels ofinflammatory cytokines in 4T1 carcinoma 24 h after dose administration.Cytokine control IL-10 PEG-mIL-10 IFNγ 36.04 68.51 98.96 IL-4 7.77 13.1340.32 IL-6 43.64 50.59 111.98 IL-10 9.94 41.62 106.16 RANK-Ligand 19.1436.13 46.08Depletion of Immune Cells

CD4+ and CD8+ T-cells were depleted by antibody-mediated elimination.250 μg of CD4- or CD8-specific antibodies were injected weekly for thispurpose. Cell depletions were verified using FACS and IHC analysis.

Depletion of CD4+ T cells in B cell deficient BALB/c mice(C.129-Igh-6^(im1Cgn)) with CD4 antibodies inhibits PEG-hIL-10 functionon tumors (Table 13).

TABLE 13 PEG-hIL-10 treatment beginning 8 days after tumor implant failsto reduce tumor size (mm³) of CT-26 colon carcinoma after CD4 depletionin B cell deficient BALB/c mice (C.129-Igh-6^(tmlCgn)). Days afterImplant 8 10 13 19 27 PBS 173 322 391 841 1979 PEG-hIL-10 184 276 251602 1332

Depletion of CD8+ T-cells completely inhibits the effect of PEG mIL-10on syngeneic tumor growth (Table 14).

TABLE 14 PEG-hIL-10 treatment beginning 8 days after tumor implant failsto reduce tumor size (mm³) of CT-26 colon carcinoma after CD8 depletionin B cell deficient BALB/c mice. Days after Implant 8 10 13 19 27 PBS151 335 584 1434 2746 PEG-hIL-10 226 575 1047 2449 4799IL-10 Dosing Frequency and Serum Trough Concentration

Murine studies were designed and performed in order to enhanceunderstanding of the pharmacokinetic parameters of IL-10 therapy and togenerate data in mice useful in optimizing the tumor treatment regimensfor recombinant human IL-10 (rhIL-10) in human subjects.

Mice were inoculated with PDV6 tumor cells, and tumors were allowed togrow for 2.5 weeks to reach 100 mm. Groups of tumor-bearing mice(n=10/group) were then treated with identical weekly doses (0.7mg/kg/week), administering 5 kDa mono-di PEGmIL-10 as a) one bolus SCinjection once a week, or b) several SC injections in divided dosesthroughout the week, including twice weekly (0.35 mg/kg), every secondday (˜0.25 mg/kg, such that the total weekly dose=0.7 mg/kg), and daily(0.1 mg/kg/day). As all mice received the same amount of drug over thecourse of a week, similar overall exposures (Area Under the Curve, AUC)were observed. The peak exposure was highest in the once-weekly dosedanimals, while the minimum drug exposure (trough) was highest in themice receiving smaller, daily doses. Surprisingly, as indicated in Table15, animals dosed daily exhibited the highest anti-tumor efficacy,indicating that the serum trough exposure was important for anti-tumorfunction, while the influence of the peak exposure on anti-tumorfunction was not determinative.

TABLE 15 Dosing Schedule Tumor Size (mm³) Control 813.9522 Daily 43.196Every second day 170.186 Bi-weekly 347.315 Weekly 425.572

The required serum trough concentration was further explored in twotumor models: PDV6 tumors in C57BL/6 mice and CT26 colon cancer cells inBalb/C mice. Utilizing standard procedures, mice were allowed to grow to100 mm³, and treatment was then initiated with administration of 5 kDamono-di PEGmIL-10 for 4 weeks. Thereafter, the serum troughconcentrations of IL-10 were measured in tumor-bearing mice receivingdifferent treatment schedules. The IL-10 serum trough concentrationswere then correlated with the resulting tumor size. As indicated inTable 16, mice with a serum trough of IL-10 over 1 ng/mL hadconsistently small tumors and rejected their tumors.

TABLE 16 IL-10 IL-10 Tumor serum trough serum trough Tumor size weightRange [pg/ml] (Mean) [pg/ml] (Mean) [mm3] (Mean) [g] 30-73 47 846 1.1105-246 164 610 0.7 250-629 433 570 0.6 1155-2095 1619 148 0.2

To confirm the critical trough concentration in human cells, hIL-10 wasadded at increasing concentrations to cultures of human peripheral bloodmonocyte cells (PBMCs). The PBMC cultures were left untreated orstimulated with lipopolysaccharide (LPS). IL-10 is known to inhibit theLPS-mediated activation of PBMCs. Activity was measured as the secretionof the chemokine MCP-1. Both LPS and IL-10 induce the secretion ofMCP-1, but inhibit each others' activity in inducing the chemokine. Atconcentrations of 1 ng/mL and above, IL-10 increased the secretion ofMCP-1 in the absence of LPS (FIG. 2A). In contrast, in PBMCs stimulatedwith LPS, addition of IL-10 at a concentration of 1 ng/mL significantlyinhibited the secretion of MCP-1 (FIG. 2B). This confirmed thebiological activity of IL-10 for both the induction and the inhibitionof respective biological processes.

Effect of IL-10 on Cytokines and Cholesterol in Human Subjects

Determination of Serum IL-10 Concentrations in Human Subjects.

Human volunteers were administered the desired amount of rhIL-10 SC orIV, and whole blood samples were drawn into heparinanticoagulant-containing vessels at desired time(s) post-administration.Serum rhIL-10 or PEG-rhIL-10 concentrations were determined using astandard sandwich enzyme-linked immune absorbent assay (ELISA) kit.Typically, the ELISA assay was determined to be selective, linear andreproducible in the concentration range of 0.1 to 10 ng/mL, and thelimit of quantitation (LOO) was 0.1 ng/mL. Serum samples were alsoanalyzed by an ELISA for the presence of antibodies that bind hIL-10. Inaddition, selected serum samples were analyzed using a validatedbioassay comprising the mouse mast cell line MC9; this cell lineproliferates in response to IL-10. The bioassay was used to determinethe bioactivity of GMP-produced rHuIL-10 and PEG-rHuIL-10 and todetermine the biological activity of the IL-10 in patient serum.Typically, ELISA and Bioassay determinations of IL-10 concentration andactivity revealed corresponding values.

Determination of TNFα and IL-1β Concentrations in Human Subjects.

IL-10 has anti-inflammatory function in patients suffering from chronicinflammatory diseases, and TNFα and IL-1β represent the key inflammatorycytokines released in such diseases. TNFα and IL-1β concentrations weredetermined in blood samples obtained from human subjects. Typically, 3mL of venous blood was aseptically collected just prior to SC or IVadministration (0 hour) of rhIL-10 and at 0.5, 2, 3, 4, 6, 8, 12, 16,24, 48, 72 and 96 hours post-dose. The samples were subjected to a wholeblood cytokine release assay in the presence of LPS and ananticoagulant, and TNFα and IL-1β concentrations were measured with anELISA assay. LPS stimulated the release of TNFα and IL-1β from bloodcells.

In samples collected 0.5-12 hours after individuals were dosed IV withrHuIL-10, the release of TNFα and IL-1β was inhibited. In samplescollected from individuals dosed SC with rHuIL-10, the release of TNFαand IL-1β was inhibited from 0.5 hours to 24 hours. The serumconcentration of rHuIL-10 in those human subjects was determined byELISA. The inhibition of TNFα and IL-1β correlated with the serumconcentration of rHuIL-10. The serum concentration of rHuIL-10 increasedafter dosing and remained elevated for 48 hours. However, the release ofTNFα and IL-1β was inhibited only as long as the concentration ofrHuIL-10 was at or above 0.2 ng/mL; the release of TNFα and IL-1β wasnot inhibited when the concentration was below 0.1 ng/mL. After 12 hfollowing IV dosing of rHuL-10 and after 24 h following SC dosing, theserum concentration dropped below 0.2 ng/mL and the release of TNFα andIL-1βP was observed. These data indicate that it is necessary to achievean IL-10 serum trough concentration at or above 0.2 ng/mL in or toobserve an anti-inflammatory function in patients suffering from chronicinflammatory diseases.

Determination of INFγ and Cholesterol Modulation by PEG-IL-10 in HumanCancer Patients.

IL-10 induces IFNγ in CD8+ T cells, and IFNγ induction is essential forIL-10-mediated tumor rejection in mice. IFNγ-deficient mice failed toreject their tumors when treated with PEG-rmIL-10 at concentrationsinducing tumor resolution in control mice (data not shown). IFNγ wastherefore measured in the serum of patients treated with PEG-rhIL-10.

After education regarding appropriate administration techniques, cancerpatients self-injected PEG-rhIL-10 SC daily at various doses. SerumIL-10 concentrations were determined using a sandwich ELISA aspreviously described. IFNγ was measured using a Luminex bead assay(Luminex Corp.; Austin, Tex.) in serum samples taken prior to the firstdose or after 28 days of dosing.

As indicated in Table 17, patients receiving 1 μg/kg PEG-IL-10 had serumtrough levels between ˜0.4 and ˜1.1 ng/mL IL-10, while patientsreceiving 2.5 μg/kg PEG-IL-10 doses had serum trough levels between ˜0.4and ˜2.6 ng/mL of IL-10.

IFNγ signals primarily through the Jak-Stat pathway. Jak-Stat signalinginvolves sequential receptor recruitment and activation of members ofthe Janus family of kinases (Jaks: Jaks 1-3 and Tyk2) and the Stats(Stats 1-6, including Stat5a and Stat5b) to control transcription oftarget genes via specific response elements. As this signaling mechanismis a characteristic of many members of the cytokine receptorsuperfamily, IFNγ-induced Jak-Stat signaling is the current paradigm forclass II cytokine receptor signal transduction. As indicated in Table17, patients having serum trough levels of 1 ng/mL or greater showed aninduction of IFNγ in the serum, while patients who had serum troughlevels below 1 ng/mL failed to show induction of IFNγ. Referring toTable 17, IFNγ induction is defined as a value greater than 1.

TABLE 17 Patient 01 02 03 04 05 Dose (μg/kg PEG- 1 1 2.5 2.5 2.5rHuIL-10) Serum Trough (ng/mL) 0.392 1.11 2.64 0.42 1.84 IFNγ Induction0.55 1.35 2.4 0.97 11.2

These data indicate that it is necessary to achieve an IL-10 serumtrough concentration at or above 1 ng/mL in or to observe a therapeuticeffect in the cancer/tumor setting. Importantly, the serum troughconcentration was the determining factor for TFNγ induction, not thedose level.

Cholesterol was measured in serum samples drawn from cancer patientsprior to administration of PEG-rhIL-10 or after one week of daily SCdosing (1 μg/kg; 2.5 μg/kg; or 5 g/kg; n=3-4 patients/dose). Referringto Table 18, patients receiving 1 μg/kg achieved an average daily serumcholesterol concentration of 0.4 ng/mL and had a 7.8% reduction incholesterol; the patient receiving 2.5 μg/kg achieved an average dailyserum cholesterol concentration of 1 ng/mL and had a 19% reduction incholesterol and the patient receiving 5 μg/kg achieved an average serumtrough cholesterol concentration of 2 ng/mL and had a 38% reduction incholesterol. Thus, each of the dosing regimens resulted in atherapeutically relevant reduction in serum cholesterol, indicating thataverage IL-10 serum trough concentrations of approximately 0.2 ng/mL to0.4 ng/mL were efficacious.

TABLE 18 Dose 1 ug/kg 2.5 5 n 4 4 3 Avg. Serum Trough 0.4 1.8 3.6 (day15) Avg. Cholesterol 7.8% 20% 37% Reduction (1 week)

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of treating a solid tumor in a humansubject, comprising: administering to the human subject atherapeutically effective amount of a PEG-IL-10 agent; wherein thePEG-IL-10 agent is mature human PEG-IL-10, is recombinantly made inbacteria, and is a mixture of mono-pegylated and di-pegylated IL-10,wherein the amount of the PEG-IL-10 agent administered is 5-20 μg/kg,and wherein the PEG-IL-10 agent is dosed daily.
 2. The method of claim1, wherein the PEG-IL-10 agent comprises at least one PEG moleculecovalently attached to at least one amino acid residue of at least onesubunit of mature human IL-10.
 3. The method of claim 1, wherein the PEGcomponent of the PEG-IL-10 agent has a molecular mass from about 5 kDato about 20 kDa.
 4. The method of claim 1, wherein said administering issubcutaneous.
 5. The method of claim 1, wherein the solid tumor is coloncancer, melanoma, squamous cell carcinoma, or lymphoma.
 6. The method ofclaim 1, wherein the solid tumor is a tumor of the pancreas.
 7. Themethod of claim 1, wherein the solid tumor is a tumor of the lung. 8.The method of claim 1, wherein the mixture of mono-pegylated anddi-pegylated IL-10 is in a ratio of 1:1.
 9. The method of claim 1,wherein the amount of the PEG-IL-10 agent administered is 5 μg/kg. 10.The method of claim 1, wherein the amount of the PEG-IL-10 agentadministered is 10 μg/kg.
 11. The method of claim 1, wherein the amountof the PEG-IL-10 agent administered is 20 μg/kg.
 12. The method of anyone of claims 1, 2, 3, 4, and 5-11, wherein the PEG-IL-10 agent isadministered in combination with 5-fluorouracil, folinic acid, and aplatinum coordination complex.